Microporation of Tissue for Delivery of Bioactive Agents

ABSTRACT

A method of enhancing the permeability of a biological membrane, including the skin or mucosa of an animal or the outer layer of a plant to a permeant is described utilizing microporation of selected depth and optionally one or more of sonic, electromagnetic, mechanical and thermal energy and a chemical enhancer. Microporation is accomplished to form a micropore of selected depth in the biological membrane and the porated site is contacted with the permeant. Additional permeation enhancement measures may be applied to the site to enhance both the flux rate of the permeant into the organism through the micropores as well as into targeted tissues within the organism.

BACKGROUND OF THE INVENTION

This invention relates generally to the field of transmembrane deliveryof drugs or bioactive molecules to an organism. More particularly, thisinvention relates to a minimally invasive to non-invasive method ofincreasing the permeability of the skin, mucosal membrane or outer layerof a plant through microporation of this biological membrane, which canbe combined with sonic, electromagnetic, and thermal energy, chemicalpermeation enhancers, pressure, and the like for selectively enhancingflux rate of bioactive molecules into the organism and, once in theorganism, into selected regions of the tissues therein.

The stratum corneum is chiefly responsible for the well known barrierproperties of skin. Thus, it is this layer that presents the greatestbarrier to transdermal flux of drugs or other molecules into the bodyand of analytes out of the body. The stratum corneum, the outer hornylayer of the skin, is a complex structure of compact keratinized cellremnants separated by lipid domains. Compared to the oral or gastricmucosa, the stratum corneum is much less permeable to molecules eitherexternal or internal to the body. The stratum corneum is formed fromkeratinocytes, which comprise the majority of epidermal cells, that losetheir nuclei and become corneocytes. These dead cells comprise thestratum corneum, which has a thickness of only about 10-30 μm and, asnoted above, is a very resistant waterproof membrane that protects thebody from invasion by exterior substances and the outward migration offluids and dissolved molecules. The stratum corneum is continuouslyrenewed by shedding of corneum cells during desquamination and theformation of new corneum cells by the keratinization process.

Underlying the stratum corneum is the viable cell layer of the epidermisand the dermis, or connective tissue layer. These layers together makeup the skin. Microporation of these underlying layers (the viable celllayer and dermis) has not previously been used but may enhancetransdermal flux. Deep to the dermis are the underlying structures ofthe body, including fat, muscle, bone, etc.

Microporation of the mucous membrane has not been used previously. Themucous membrane generally lacks a stratum corneum. The most superficiallayer is the epithelial layer which consists of numerous layers ofviable cells. Deep to the epithelial layer is the lamina propria, orconnective tissue layer.

Microporation of plants has been previously limited to selectapplications in individual cells in laboratory settings. Plant organismsgenerally have tough outer layers to provide resistance to the elementsand disease. Microporation of this tough outer layer of plants enablesthe delivery of substances useful for introduction into the plant suchas for conferring the desired trait to the plant or for production of adesired substance. For example, a plant may be treated such that eachcell of the plant expresses a particular and useful peptide such as ahormone or human insulin.

The flux of a drug or analyte across the biological membrane can beincreased by changing either the resistance (the diffusion coefficient)or the driving force (the gradient for diffusion). Flux may be enhancedby the use of so-called penetration or chemical enhancers. Chemicalenhancers are well known in the art and a more detailed description willfollow.

Another method of increasing the permeability of skin to drugs isiontophoresis. Iontophoresis involves the application of an externalelectric field and topical delivery of an ionized form of drug or anun-ionized drug carried with the water flux associated with iontransport (electro-osmosis): While permeation enhancement withiontophoresis has been effective, control of drug delivery andirreversible skin damage are problems associated with the technique.

Sonic energy has also been used to enhance permeability of the skin andsynthetic membranes to drugs and other molecules. Ultrasound has beendefined as mechanical pressure waves with frequencies above 20 kHz, H.Lutz et al., Manual of Ultrasound 3-12 (1984). Sonic energy is generatedby vibrating a piezoelectric crystal or other electromechanical elementby passing an alternating current through the material, R. Brucks etal., 6 Pharm. Res. 697 (1989). The use of sonic energy to increase thepermeability of the skin to drug molecules has been termed sonophoresisor phonophoresis.

Although it has been acknowledged that enhancing permeability of theskin should theoretically make it possible to transport molecules frominside the body through the skin to outside the body for collection ormonitoring, practicable methods have not been disclosed. U.S. Pat. No.5,139,023 to Stanley et al. discloses an apparatus and method fornoninvasive blood glucose monitoring. In this invention, chemicalpermeation enhancers are used to increase the permeability of mucosaltissue or skin to glucose. Glucose then passively diffuses through themucosal tissue or skin and is captured in a receiving medium. The amountof glucose in the receiving medium is measured and correlated todetermine the blood glucose level. However; as taught in Stanley et al.,this method is much more efficient when used on mucosal tissue, such asbuccal tissue, which results in detectable amounts of glucose beingcollected in the receiving medium after a lag time of about 10-20minutes. However, the method taught by Stanley et al. results in anextremely long lag time, ranging from 2 to 24 hours depending on thechemical enhancer composition used, before detectable amounts of glucosecan be detected diffusing through human skin (heat-separated epidermis)in vitro. These long lag times may be attributed to the length of timerequired for the chemical permeation enhancers to passively diffusethrough the skin and to enhance the permeability of the barrier stratumcorneum, as well as the length of time required for the glucose topassively diffuse out through the skin. Thus, Stanley et al. clearlydoes not teach a method for transporting blood glucose or other analytesnon-invasively through the skin in a manner that allows for rapidmonitoring, as is required for blood glucose monitoring of diabeticpatients and for many other body analytes such as blood electrolytes.

While the use of sonic energy for drug delivery is known, results havebeen largely disappointing in that enhancement of permeability has beenrelatively low. There is no consensus on the efficacy of sonic energyfor increasing drug flux across the skin. While some studies report thesuccess of sonophoresis, J. Davick et al., 68 Phys. Ther. 1672 (1988);J. Griffin et al., 47 Phys. Ther. 594 (1967); J. Griffin & J.Touchstone, 42 Am. J. Phys. Med, 77 (1963); J. Griffin et al., 44 Am. J.Phys. Med. 20 (1965); D. Levy et al., 83 J. Clin. Invest. 2074); D.Bommannan et al., 9 Pharm. Res. 559 (1992), others have obtainednegative results, H. Benson et al., 69 Phys. Ther. 113 (1988); J.McElnay et al., 20 Br. J. Clin. Pharmacol. 4221 (1985); H. Pratzel etal., 13 J. Rheumatol. 1122 (1986). Systems in which rodent skin wereemployed showed the most promising results, whereas systems in whichhuman skin was employed have generally shown disappointing results. Itis well known to those skilled in the art that rodent skin is much morepermeable than human skin, and consequently the above results do notteach one skilled in the art how to effectively utilize sonophoresis asapplied to transdermal delivery and/or monitoring through human skin.

A significant improvement in the use of ultrasonic energy in themonitoring of analytes and also in the delivery of drugs to the body isdisclosed and claimed in copending applications Ser. No. 08/152,442filed Nov. 15, 1993, now U.S. Pat. No. 5,458,140, and Ser. No.08/152,174 filed Dec. 8, 1993, now U.S. Pat. No. 5,445,611, both ofwhich are incorporated herein by reference. In these inventions, thetransdermal sampling of an analyte or the transdermal delivery of drugs,is accomplished through the use of sonic energy that is modulated inintensity, phase, or frequency or a combination of these parameterscoupled with the use of chemical permeation enhancers. Also disclosed isthe use of sonic energy, optionally with modulations of frequency,intensity, and/or phase, to controllably push and/or pump moleculesthrough the stratum corneum via perforations introduced by needlepuncture, hydraulic jet, laser, electroporation, or other methods.

The formation of micropores (i.e. microporation) in the stratum corneumto enhance the delivery of drugs has been the subject of various studiesand has resulted in the issuance of patents for such techniques.

Jacques et al., 88 J. Invest. Dermatol. 88-93 (1987), teaches a methodof administering a drug by ablating the stratum corneum of a region ofthe skin using pulsed laser light of wavelength, pulse length, pulseenergy, pulse number, and pulse repetition rate sufficient to ablate thestratum corneum without significantly damaging the underlying epidermisand then applying the drug to the region of ablation. This work resultedin the issuance of U.S. Pat. No. 4,775,361 to Jacques et al. Theablation of skin through the use of ultraviolet-laser irradiation wasearlier reported by Lane et al., 121 Arch. Dermatol. 609-617 (1985).Jacques et al. is restricted to use of few wavelengths of light andexpensive lasers.

Tankovich, U.S. Pat. No. 5,165,418 (hereinafter, “Tankovich '418”),discloses a method of obtaining a blood sample by irradiating human oranimal skin with one or more laser pulses of sufficient energy to causethe vaporization of skin tissue so as to produce a hole in the skinextending through the epidermis and to sever at least one blood vessel,causing a quantity of blood to be expelled through the hole such that itcan be collected. Tankovich '418 thus is inadequate for noninvasive orminimally invasive permeabilization of the stratum corneum such that adrug can be delivered to the body or an analyte from the body can beanalyzed.

Tankovich et al., U.S. Pat. No. 5,423,803 (hereinafter, “Tankovich'803”) discloses a method of laser removal of superficial epidermal skincells in human skin for cosmetic applications. The method comprisesapplying a light-absorbing “contaminant” to the outer layers of theepidermis and forcing some of this contaminant into or through theintercellular spaces in the stratum corneum, and illuminating theinfiltrated skin with pulses of laser light of sufficient intensity thatthe amount of energy absorbed by the contaminant will cause thecontaminant to explode with sufficient energy to tear off some of theepidermal skin cells. Tankovich '803 further teaches that there shouldbe high absorption of energy by the contaminant at the wavelength of thelaser beam, that the laser beam must be a pulsed beam of less than 1 μsduration, that the contaminant must be forced into or through the upperlayers of the epidermis, and that the contaminant must explode withsufficient energy to tear off epidermal cells upon absorption of thelaser energy. This invention also fails to disclose or suggest a methodof drug delivery or analyte collection.

Raven et al., WO 92/00106, describes a method of selectively removingunhealthy tissue from a body by administering to a selected tissue acompound that is highly absorbent of infrared radiation of wavelength750-860 nm and irradiating the region with corresponding infraredradiation at a power sufficient to cause thermal vaporization of thetissue to which the compound was administered but insufficient to causevaporization of tissue to which the compound had not been administered.The absorbent compound should be soluble in water or serum, such asindocyanine green, chlorophyll, porphyrins, heme-containing compounds,or compounds containing a polyene structure, and power levels are in therange of 50-1000 W/cm² or even higher.

Konig et al., DD 259351, teaches a process for thermal treatment oftumor tissue that comprises depositing a medium in the tumor tissue thatabsorbs radiation in the red and/or near red infrared spectral region,and irradiating the infiltrated tissue with an appropriate wavelength oflaser light. Absorbing media can include methylene blue, reducedporphyrin or its aggregates, and phthalocyanine blue. Methylene blue,which strongly absorbs at 600-700 nm, and a krypton laser emitting at647 and 676 nm are exemplified. The power level should be at least 200mW/cm².

It has been shown that by stripping the stratum corneum from a smallarea of the skin with repeated application and removal of cellophanetape to the same location one can easily collect arbitrary quantities ofinterstitial fluid, which can then be assayed for a number of analytesof interest. Similarly, the ‘tape-stripped’ skin has also been shown tobe permeable to the transdermal delivery of compounds into the body.Unfortunately, ‘tape-stripping’ leaves a open sore which takes weeks toheal, and for this, as well as other reasons, is not considered as anacceptable practice for enhancing transcutaneous transport in wideapplications.

As discussed above, it has been shown that pulsed lasers, such as theexcimer laser operating at 193 nm, the erbium laser operating near 2.9μm or the CO₂ laser operating at 10.2 μm, can be used to effectivelyablate small holes in the human stratum corneum. These laser ablationtechniques offer the potential for a selective and potentiallynon-traumatic method for opening a delivery and/or sampling hole throughthe stratum corneum. However, due to the prohibitively high costsassociated with these light sources, there have been no commercialproducts developed based on this concept. The presently disclosedinvention, by defining a method for directly conducting thermal energyinto or through the biological membrane with very tightly definedspatial and temporal resolution, makes it possible to produce thedesired micro-ablation of the biological membrane very low cost energysources.

In view of the foregoing problems and/or deficiencies, the developmentof a method for safely enhancing the permeability of the biologicalmembrane for minimally invasive or noninvasive monitoring of bodyanalytes in a more rapid time frame would be a significant advancementin the art. It would be another significant advancement in the art toprovide a method of minimally invasively or non-invasively enhancing thetransmembrane flux rate of a drug into a selected area of an organism.

Significant advancements in the delivery of drugs and other compoundsare being made through the use of various techniques that increase thepermeability of a biological membrane, such as the skin or mucosalmembrane. Even more promising advances have been made through techniquesfor creating micropores, as disclosed in the aforementionedapplications.

Nevertheless, it is desirable to improve upon these technologies byforming micropores at selected depths in the biological membrane and todeliver both small and large compounds, in terms of molecular weight andsize, through the micropores into the body.

BRIEF SUMMARY OF THE INVENTION

This invention provides a method for enhancing the transmembrane fluxrate of a permeant into a selected site of an organism comprising thesteps of enhancing the permeability of said selected site of theorganism to said permeant by means of (a) porating a biological membraneat said selected site by means that form a micropore in said biologicalmembrane, thereby reducing the barrier properties of said biologicalmembrane to the flux of said permeant and (b) contacting the poratedselected site with a composition comprising an effective amount of saidpermeant, whereby the transmembrane flux rate of said permeant into theorganism is enhanced.

This invention further provides the method of enhancing thetransmembrane flux rate further comprising applying to said site of saidorganism an enhancer to increase the flux of said permeant into saidorganism. The invention also provides the method wherein said enhancercomprises sonic energy, and more specifically, wherein the said sonicenergy is applied to said site at a frequency in the range of about 10Hz to 1000 MHz, and wherein said sonic energy is modulated by means of amember selected from the group consisting of frequency modulation,amplitude modulation, phase modulation, and combinations thereof.Alternatively, the said enhancer comprises an electromagnetic field,and, more specifically, iontophoresis or a magnetic field, or amechanical force, chemical enhancer, or thermal enhancer. Additionally,the invention further provides a method wherein any of the methods ofsonic, electromagnetic, mechanical, thermal, or chemical enhancement maybe applied in any combination thereof to increase the transmembrane fluxrate of said permeant into or through said micropore.

This invention also provides a method of further enhancing thetransmembrane flux rate with an enhancer, wherein said enhancers at saidsite are applied so as to increase the flux rate of the permeant intotissues surrounding the micropore. The said enhancer can comprise sonicenergy. Furthermore, the said sonic energy is applied to said site at afrequency in the range of about 10 Hz to 1000 MHz, wherein said sonicenergy is modulated by means of a member selected from the groupconsisting of frequency modulation, amplitude modulation, phasemodulation, and combinations thereof. Alternatively, the said enhancercomprises sonic or thermal energy, electroporation, iontophoresis,chemical enhancers, mechanical force, or a magnetic field, or anycombination thereof.

The invention further includes the method of enhancing the transmembraneflux rate of a permeant further comprising applying to said site of saidorganism an enhancer, wherein any of the methods of methods of sonic orthermal energy, electroporation, iontophoresis, chemical enhancers,mechanical force, or a magnetic field may be applied in any combinationthereof further comprising the method of combining sonic or thermalenergy, electroporation, iontophoresis, chemical enhancers, mechanicalforce, or a magnetic field to increase the flux rate of the permeantinto tissues surrounding the micropore.

The invention also includes the method of further enhancing thetranmembrane flux rate within and beneath the outer layer wherein saidporating of said biological membrane in said site is accomplished bymeans selected from the group consisting of (a) ablating the biologicalmembrane by contacting said site, up to about 1000 μm across, of saidbiological membrane with a heat source such that a micropore is formedin said biological membrane at said site; (b) puncturing said biologicalmembrane with a micro-lancet calibrated to form a micropore of up toabout 1000 μm in diameter; (c) ablating the biological membrane by abeam of sonic energy onto said biological membrane up to about 1000 μmin diameter; (d) hydraulically puncturing said biological membrane witha high pressure jet of fluid to form a micropore of up to about 1000 μmin diameter and (e) puncturing said biological membrane with shortpulses of electricity to form a micropore of up to about 1000 μm indiameter. Further, the invention includes the method wherein saidporating is accomplished by contacting said site, up to about 1000 μmacross, with a heat source to conductively transfer an effective amountof thermal energy to said site such that the temperature of some of thewater and other vaporizable substances in said site is elevated abovetheir vaporization point creating a micropore to a selected depth in thebiological membrane at said site or wherein said porating isaccomplished by contacting said site, up to about 1000 μm across, with aheat source to conductively transfer an effective amount of thermalenergy to said site such that the temperature of some of the tissue atsaid site is elevated to the point where thermal decomposition occurscreating a micropore to a selected depth in the biological membrane atsaid site. Additionally, the invention includes the method of poratingsaid biological membrane in said site further comprising treating atleast said site with an effective amount of a substance that exhibitssufficient absorption over the emission range of a pulsed light sourceand focusing the output of a series of pulses from said pulsed lightsource onto said substance such that said substance is heatedsufficiently to conductively transfer an effective amount of thermalenergy to said biological membrane to elevate the temperature to therebycreate a micropore. The invention also includes the method wherein saidpulsed light source emits at a wavelength that is not significantlyabsorbed by said biological membrane. The invention further provides themethod wherein said pulsed light source is a laser diode emitting in therange of about 630 to 1550 nm, wherein said pulsed light source is alaser diode pumped optical parametric oscillator emitting in the rangeof about 700 and 3000 nm, wherein said pulsed light source is a memberselected from the group consisting of arc lamps, incandescent lamps, andlight emitting diodes. The invention also includes the method furthercomprising providing a sensing system for determining when the microporein the biological membrane has reached the desired dimensions, includingwidth, length, and depth, and, further, wherein said sensing systemcomprises light collection means for receiving light reflected from saidsite and focusing said reflected light on a detector for receiving saidlight and sending a signal to a controller wherein said signal indicatesa quality of said light, and a controller coupled to said detector andto said light source for receiving said signal and for shutting off saidlight source when a preselected signal is received, or, alternatively,an electrical impedance measuring system which can detect the changes inthe impedance of the biological membrane at different depths into theorganism as the micropore is formed.

The invention also provides the method of enhancing the tranmembraneflux rate within and beneath the outer layer further comprising coolingsaid site and adjacent tissues such that said site and adjacent tissuesare in a cooled condition. The said cooling means comprises a Peltierdevice.

The invention also includes the method of enhancing the transmembraneflux within and beneath the outer layer further comprising, prior toporating said site, illuminating at least said site with light such thatsaid site is sterilized.

This invention also includes the method of enhancing the transmembraneflux within and beneath the outer layer further comprising contactingsaid site with a solid element, wherein said solid element functions asa heat source to conductively transfer an effective amount of thermalenergy to said biological membrane to elevate the temperature to therebycreate a micropore. Further, said heat source is constructed to modulatethe temperature of said site to greater than 100° C. within about 10nanoseconds to 50 milliseconds and then returning the temperature ofsaid site to approximately ambient temperature within about 1millisecond to 50 milliseconds and wherein a cycle of raising thetemperature and returning to ambient temperature is repeated one or moretimes effective for porating the biological membrane to the desireddepth. The invention further includes the method of using a heat sourcewherein said returning to approximately ambient temperature of said siteis carried out by withdrawing said heat source from contact with saidsite and wherein the modulation parameters are selected to reducesensation to the animal subject.

The invention includes the method for enhancing transmembrane flux ratesusing a heat source and sensing system further comprising providingmeans for monitoring electrical impedance between said solid element andsaid organism through said site and adjacent tissues and means foradvancing the position of said solid element such that as said porationoccurs with a concomitant change in impedance, said advancing meansadvances the solid element such that the solid element is in contactwith said site during heating of the solid element, until the selectedimpedance is obtained. Further, the invention includes this methodfurther comprising means for withdrawing said solid element from contactwith said site wherein said monitoring means is capable of detecting achange in impedance associated with contacting a selected layerunderlying the surface of said site and sending a signal to saidwithdrawing means to withdrawn said solid element from contact with saidsite.

The method of enhancing the transmembrane flux rate using a solidelement wherein said solid element is heated by delivering an electricalcurrent through an ohmic heating element and, further, wherein saidsolid element is formed such that it contains an electrically conductivecomponent and the temperature of said solid element is modulated bypassing a modulated electrical current through said conductive element.Additionally, the invention includes the method wherein said solidelement is positioned in a modulatable magnetic field wherein energizingthe magnetic field produces electrical eddy currents sufficient to heatthe solid element.

The invention also includes the method of enhancing the transmembraneflux rate wherein said porating is accomplished by puncturing said sitewith a micro-lancet calibrated to form a micropore of up to about 1000μm in diameter, by a beam of sonic energy directed onto said site toform a micropore of up to about 1000 μm in diameter, by hydraulicallypuncturing said biological membrane with a high pressure jet of fluid toform a micropore of up to about 1000 μm in diameter, or, alternatively,by puncturing said biological membrane with short pulses of electricityto form a micropore of up to about 1000 μm in diameter.

The invention further comprises the method of enhancing thetransmembrane flux rate of a permeant wherein said permeant comprises anucleic acid. More specifically, the invention includes the methodwherein said nucleic acid comprises DNA or wherein the nucleic acidcomprises RNA.

The invention further includes the method of enhancing the transmembraneflux rate of a permeant wherein the micropore in the biological membraneextends into a portion of the outer layer of the biological membraneranging from 1 to 30 microns in depth, extends through the outer layerof the biological membrane ranging from 10 to 200 microns in depth,extends into the connective tissue layer of the biological membraneranging from 100 to 5000 microns in depth, or extends through theconnective tissue layer of the biological membrane ranging from 1000 to10000 microns in depth.

The invention further includes the method of enhancing the transmembraneflux rate of a permeant, wherein the micropore penetrates the biologicalmembrane to a depth determined to facilitate desired activity of theselected permeant.

The invention further includes the method of enhancing the transmembraneflux rate of a permeant wherein the permeant comprises a polypeptide,including wherein the polypeptide is a protein or a peptide, and furtherincluding wherein the peptide comprises insulin or a releasing factor; acarbohydrate, including wherein the carbohydrate comprises a heparin; ananalgesic, including wherein the analgesic comprises an opiate; avaccine; or a steroid.

The invention further includes the method of enhancing the transmembraneflux rate of a permeant wherein the permeant is associated with acarrier. The invention further includes the method wherein the carriercomprises liposomes; lipid complexes; microparticles; or polyethyleneglycol compounds. More specifically, the invention further includes themethod wherein the permeant is a vaccine in combination with the methodwherein the permeant is associated with a carrier.

The invention further includes the method of enhancing the transmembraneflux rate of a permeant wherein the permeant comprises a substance whichhas the ability to change its detectable response to a stimulus when inthe proximity of an analyte present in the organism.

An object of the invention is to provide a method for controllingtransmembrane flux rates of drugs or other molecules into the body and,if desired, into the bloodstream through minute perforations in thebiological membrane, including stratum corneum or other layers of theskin or in the mucosa or outer layers of a plant.

It is still another object of the invention to provide a method ofdelivering drugs into the body through micropores in the biologicalmembrane in combination with sonic energy, permeation enhancers,pressure gradients, electromagnetic energy, thermal energy, and thelike.

An object of the invention is to minimize the barrier properties of thebiological membrane using poration to controllably collect analytes fromwithin the body through perforations in the biological membrane toenable the monitoring of these analytes.

It is also an object of the invention to provide a method of monitoringselected analytes in the body through micropores in the biologicalmembrane in combination with sonic energy, permeation enhancers,pressure gradients, electromagnetic energy, mechanical energy, thermalenergy, and the like.

These and other objects may be accomplished by providing a method formonitoring the concentration of an analyte in an individual's bodycomprising the steps of enhancing the permeability of the biologicalmembrane of a selected area of the individual's body surface to theanalyte by means of.

(a) porating the biological membrane of the selected area by means thatform a micropore in the biological membrane optionally without causingserious damage to the underlying tissues, thereby reducing the barrierproperties of the biological membrane to, the withdrawal of the analyte;

(b) collecting a selected amount of the analyte; and

(c) quantitating the analyte collected.

In one preferred embodiment, the method further comprises applying sonicenergy to the porated selected area at a frequency in the range of about5 kHz to 100 MHz, wherein the sonic energy is modulated by means of amember selected from the group consisting of frequency modulation,amplitude modulation, phase modulation, and combinations thereof. Inanother preferred embodiment, the method comprises contacting theselected area of the individual's body with a chemical enhancer with theapplication of electromagnetic, thermal, mechanical, or sonic energy tofurther enhance analyte withdrawal.

Porating of the biological membrane is accomplished by means selectedfrom the group consisting of (a) ablating the biological membrane bycontacting a selected area, up to about 1000 μm across, of thebiological membrane with a heat source such that the temperature oftissue-bound water and other vaporizable substances in the selected areais elevated above the vaporization point of the water and othervaporizable substances thereby removing the biological membrane in theselected area; (b) puncturing the biological membrane with amicro-lancet calibrated to form a micropore of up to about 1000 μm indiameter; (c) ablating the biological membrane by focusing a tightlyfocused beam of sonic energy onto the stratum corneum; (d) hydraulicallypuncturing the biological membrane with a high pressure jet of fluid toform a micropore of up to about 1000 μm in diameter and (e) puncturingthe biological membrane with short pulses of electricity to form amicropore of up to about 1000 μm in diameter.

One preferred embodiment of thermally ablating the biological membranecomprises treating at least the selected area with an effective amountof a dye that exhibits strong absorption over the emission range of apulsed light source and focusing the output of a series of pulses fromthe pulsed light source onto the dye such that the dye is heatedsufficiently to conductively transfer heat to the stratum corneum toelevate the temperature of tissue-bound water and other vaporizablesubstances in the selected area above the vaporization point of thewater and other vaporizable substances. Preferably, the pulsed lightsource emits at a wavelength that is not significantly absorbed by skin.For example, the pulsed light source can be a laser diode emitting inthe range of about 630 to 1550 nm, a laser diode pumped opticalparametric oscillator emitting in the range of about 700 and 3000 nm, ora member to selected from the group consisting of arc lamps,incandescent lamps, and light emitting diodes. A sensing system fordetermining when the barrier properties of the stratum corneum have beensurmounted can also be provided. One preferred sensing system compriseslight collection means for receiving light reflected from the selectedarea and focusing the reflected light on a photodiode, a photodiode forreceiving the focused light and sending a signal to a controller whereinthe signal indicates a quality of the reflected light, and a controllercoupled to the photodiode and to the pulsed light source for receivingthe signal and for shutting off the pulsed light source when apreselected signal is received.

In another preferred embodiment, the method further comprises coolingthe selected area of biological membrane and adjacent tissues withcooling means such that said selected area and adjacent tissues are in aselected cooled, steady state, condition prior to, during, and/or afterporation.

In still another preferred embodiment, the method comprises ablating thebiological membrane such that interstitial fluid exudes from themicropores, collecting the interstitial fluid, and analyzing the analytein the collected interstitial fluid. After the interstitial fluid iscollected, the micropore can be sealed by applying an effective amountof energy from the laser diode or other light source such thatinterstitial fluid remaining in the micropore is caused to coagulate.Preferably, vacuum is applied to the porated selected area to enhancecollection of interstitial fluid.

In yet another preferred embodiment, the method comprises, prior toporating the biological membrane, illuminating at least the selectedarea with light such that the selected area illuminated with the lightis sterilized.

Another preferred method of porating the biological membrane comprisescontacting the selected area with a solid element such that thetemperature of the selected area is raised from ambient temperature togreater than 100° C. within about 10 nanoseconds to 50 ms and thenreturning the temperature of the selected area to approximately ambientskin temperature within about 1 to 50 ms, wherein this cycle of raisingthe temperature and returning to approximately ambient temperature isrepeated a number of time effective for reducing the barrier propertiesof the biological membrane. Preferably, the step of returning toapproximately ambient temperature is carried out by withdrawing thesolid element from contact with the biological membrane. It is alsopreferred to provide means for monitoring electrical impedance betweenthe solid element and the body through the selected area of biologicalmembrane and adjacent tissues and means for advancing the position ofthe solid element such that as the ablation occurs with a concomitantreduction in resistance, the advancing means advances the solid elementsuch that the solid element is in contact with the biological membraneduring heating of the solid element. Further, it is also preferred toprovide means for withdrawing the solid element from contact with thebiological membrane, wherein the monitoring means is capable ofdetecting a change in impedance associated with contacting a layerunderlying the biological membrane or a layer thereof and sending asignal to the withdrawing means to withdrawn the solid element fromcontact with the biological membrane. The solid element can be heated byan ohmic heating element, can have a current loop having a highresistance point wherein the temperature of the high resistance point ismodulated by passing a modulated electrical current through said currentloop to effect the heating, or can be positioned in a modulatablealternating magnetic field of an excitation coil such that energizingthe excitation coil with alternating current produces eddy currentssufficient to heat the solid element by internal ohmic losses.

A method for enhancing the transmembrane flux rate of an active permeantinto a selected area of a body comprising the steps of enhancing thepermeability of the biological membrane layer of the selected area ofthe body surface to the active permeant by means of

(a) porating the biological membrane of the selected area by means thatform a micropore in the biological membrane optionally without causingserious damage to the underlying tissues and thereby reducing thebarrier properties of the biological membrane to the flux of the activepermeant; and

(b) contacting the porated selected area with a composition comprisingan effective amount of the permeant such that the flux of the permeantinto the body is enhanced.

In a preferred embodiment, the method further comprises applying energyto the porated selected area for a time and at an intensity and afrequency effective to create a fluid streaming effect and therebyenhance the transmembrane flux rate of the permeant into the body.

A method is also provided for applying a tattoo to a selected area ofskin on an individual's body surface comprising the steps of:

(a) porating the stratum corneum of the selected area by means that forma micropore in the stratum corneum optionally without causing seriousdamage to the underlying tissues and thereby reduce the barrierproperties of the stratum corneum to the flux of a permeant; and

(b) contacting the porated selected area with a composition comprisingan effective amount of a tattooing ink as a permeant such that the fluxof said ink into the body is enhanced.

A method is still further provided for reducing a temporal delay indiffusion of an analyte from blood of an individual to said individual'sinterstitial fluid in a selected area of biological membrane comprisingapplying means for cooling to said selected area of skin.

A method is yet further provided for reducing evaporation ofinterstitial fluid and the vapor pressure thereof, wherein saidinterstitial fluid is being collected from a micropore in a selectedarea of the biological membrane of an individual, comprising applyingmeans for cooling to said selected area of biological membrane.

In accordance with still further embodiments, the present invention isdirected to a method for delivering bioactive agents into the bodythrough micropores formed at selected depths in a biological membrane,such as the skin or mucous membrane or outer layer of a plant. Themethod involves porating an outer layer of the biological membranethrough any of the poration techniques known in the art, but to asufficient and desired depth into or through the biological membrane,and contacting the porated site with an effective quantity of thebioactive agent of low or high molecular weight and size. This processcan be enhanced by applying further permeation enhancement measureseither before, during or after the bioactive agent is delivered. Forexample, sonic energy, iontophoresis, magnetic fields, electroporation,chemical permeation enhancer; osmotic pressure and atmospheric pressuremeasures may be applied to the porated site to enhance the permeabilityof layers beneath the outer layer of the biological membrane.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows a schematic representation of a system for delivering laserdiode light and monitoring the progress of poration.

FIG. 2 shows a schematic representation of a closed-loop feedback systemfor monitoring poration.

FIG. 3A shows a schematic representation of an optical poration systemcomprising a cooling device.

FIG. 3B shows a top view of a schematic representation of anillustrative cooling device according to FIG. 3B.

FIG. 4 shows a schematic representation of an ohmic heating device witha mechanical actuator.

FIG. 5 shows a schematic representation of a high resistance currentloop heating device.

FIG. 6 shows a schematic representation of a device for modulatingheating using inductive heating.

FIG. 7 shows a schematic representation of a closed loop impedancemonitor using changes in impedance to determine the extent of poration.

FIGS. 8A-D show cross sections of human skin treated with copperphthalocyanine and then subjected, respectively, to 0, 1, 5, and 50pulses of 810 nm light with an energy density of 4000 J/cm² for a pulseperiod of 20 ms.

FIGS. 9-11 show graphic representations of temperature distributionduring simulated thermal poration events using optical poration.

FIGS. 12 and 13 show graphic representations of temperature as afunction of time in the stratum corneum and viable epidermis,respectively, during simulated thermal poration events using opticalporation.

FIGS. 14-16 show graphic representations of temperature distribution,temperature as a function of time in the stratum corneum, andtemperature as a function of time in the viable epidermis, respectively,during simulated thermal poration events using optical poration whereinthe tissue was cooled prior to poration.

FIGS. 17-19 show graphic representations of temperature distribution,temperature as a function of time in the stratum corneum, andtemperature as a function of time in the viable epidermis, respectively,during simulated thermal poration events wherein the tissue was heatedwith a hot wire.

FIGS. 20-22 show graphic representations of temperature distribution,temperature as a function of time in the stratum corneum, andtemperature as a function of time in the viable epidermis, respectively,during simulated thermal poration events wherein the tissue was heatedwith a hot wire and the tissue was cooled prior to poration.

FIGS. 23 and 24 show graphic representations of temperature distributionand temperature as a function of time in the stratum corneum,respectively, during simulated thermal poration events wherein thetissue is heated optically according to the operating parameters ofTankovich '803.

FIG. 25 shows a graphic representation of interstitial fluid (ISF;) andblood (*) glucose levels as a function of time.

FIG. 26 shows a scatter plot representation of the difference termbetween the ISF glucose and the blood glucose data of FIG. 25.

FIG. 27 shows a histogram of the relative deviation of the ISF to theblood glucose levels from FIG. 25.

FIG. 28 shows a cross section of an illustrative delivery apparatus fordelivering a drug to a selected area on an individual's skin.

FIGS. 29A-C show graphic representations of areas of skin affected bydelivery of lidocaine to selected areas where the stratum corneum isporated (FIGS. 29A-B) or not porated (FIG. 29C).

FIG. 30 shows a plot comparing the amount of interstitial fluidharvested from micropores with suction alone ( ) and with a combinationof suction and ultrasound (*).

FIGS. 31, 32, and 33 show a perspective view of an ultrasonictransducer/vacuum apparatus for harvesting interstitial fluid, a crosssection view of the same apparatus, and cross sectional schematic viewof the same apparatus, respectively.

FIGS. 34A-B show a top view of a handheld ultrasonic transducer and aside view of the spatulate end thereof, respectively.

FIG. 35 is a graphical representation showing the enhancing effects ofmicroporation in the transdermal delivery of testosterone.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

It is to be understood that this invention is not limited to“theparticular configurations, process steps, and materials disclosed hereinas such configurations, process steps, and materials may vary somewhat.It is also to be understood that the terminology employed herein is usedfor the purpose of describing particular embodiments only and is notintended to be limiting since the scope of the present invention will belimited only by the appended claims and equivalents thereof.

It must be noted that, as used herein the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to a method for delivery of “adrug” includes reference to delivery of a mixture of two or more drugs,reference to “an analyte” includes reference to one or more of suchanalytes, and reference to “a permeation enhancer” includes reference toa mixture of two or more permeation enhancers or techniques such as acombination of ultrasound and electroporation.

Thus, as used herein, the singular form may be used interchangeably withthe plural form, and vice versa, i.e. “layer” could mean layers or“layers” could mean layer.

As used herein, “including” or “includes” or the like means including,without limitation.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

As used herein “organism” means the entire animal or plant being actedupon by the methods described herein.

As used herein, “poration,” “microporation,” or any such similar termmeans the formation of a small hole or pore to a desired depth in orthrough the biological membrane, such as skin or mucous membrane, or theouter layer of an organism to lessen the barrier properties of thisbiological membrane to the passage of analytes from below the surfacefor analysis or the passage of permeants or drugs into the body forselected purposes, or for certain medical or surgical procedures. Themicroporation process referred to herein is distinguished from theopenings formed by electroporation principally by the minimum dimensionsof the micropores which shall be no smaller than 1 micron across and atleast 1 micron in depth, whereas the openings formed withelectroporation are typically only a few nanometers in any dimension.Nevertheless, electroporation is useful to facilitate uptake of selectedpermeants by the targeted tissues beneath the outer layers of anorganism after the permeant has passed through the micropores into thesedeeper layers of tissue. Preferably the hole or micropore will be nolarger than about 1 mm in diameter, and more preferably no larger thanabout 300 μm in diameter, and will extend to a selected depth, asdescribed hereinafter.

As used herein, “micropore” or “pore” means an opening formed by themicroporation method.

As used herein “ablation” means the controlled removal of material whichmay include cells or other components comprising some portion of abiological membrane or tissue caused by any of the following: kineticenergy released when some or all of the vaporizable components of suchmaterial have been heated to the point that vaporization occurs and theresulting rapid expansion of volume due to this phase change causes thismaterial, and possibly some adjacent material, to be removed from theablation site; thermal, mechanical, or sonic decomposition of some orall off the tissue at the poration site.

As used herein ablation of a tissue or puncture of a tissue may beachieved utilizing the same energy source.

As used herein, “tissue” means any component of an organism includingbut not limited to, cells, biological membranes, bone, collagen, fluidsand the like comprising some portion of the organism.

As used herein, “sonic” or “acoustic” are interchangeable and cover thefrequency space from 0.01 Hz and up.

As used herein, “ultrasonic” describes a subset of sonic comprisingfrequencies greater or equal to 20,000 Hz with no upper limit.

As used herein “puncture” or “micro-puncture” means the use ofmechanical, hydraulic, sonic, electromagnetic, or thermal means toperforate wholly or partially a biological membrane such as the skin ormucosal layers of an animal or the outer tissue layers of a plant.

To the extent that “ablation” and “puncture” accomplish the same purposeof poration, i.e. the creating a hole or pore in the biological membraneoptionally without significant damage to the underlying tissues, theseterms may be used interchangeably.

As used herein, “penetration enhancement” or “permeation enhancement”means an increase in the permeability by utilization of a permeationenhancer of a biological membrane such as the skin or mucosal or buccalmembrane or a plant's outer layer of tissue to a bioactive agent, drug,analyte, dye, stain, microparticle, microsphere, compound, or otherchemical formulation (also called “permeant”), i.e., so as to increasethe rate at which a bioactive agent, drug, analyte, stain,micro-particle, microsphere, compound, or other chemical formulationpermeates the biological membrane and facilitates the withdrawal ofanalytes out through the biological membrane or the delivery ofsubstances through the biological membrane and into the underlyingtissues. The enhanced permeation effected through the use of suchenhancers can be observed, for example, by observing diffusion of a dye,as a permeant, through animal or human skin using a diffusion apparatus.

As used herein, “penetration enhancer,” “permeation enhancer,”“enhancer,” and the like includes all substances and techniques thatincrease the flux of a permeant, analyte, or other molecule across theskin, and is limited only by functionality. In other words, all cellenvelope disordering compounds and solvents and physical techniques suchas electroporation, iontophoresis, magnetic fields, sonic energy,thermal energy, or mechanical pressure or manipulation such as a localmassaging of the site and any chemical enhancement agents are intendedto be included.

As used herein “ chemical enhancer” means a substance that increases theflux of a permeant or analyte or other substance across a biologicalmembrane and is limited only by function.

As used herein, “dye,” “stain,” and the like shall be usedinterchangeably and refer to a biologically suitable chromophore thatexhibits suitable absorption over some or all of the emission range of apulsed light source used to ablate tissues to form micropores therein.

As used herein, “transdermal” or “percutaneous” or “transmembrane” or“transmucosal” or “transbuccal” means passage of a permeant into orthrough the biological membrane or tissue to achieve effectivetherapeutic blood levels or tissue levels of a drug, or the passage of amolecule present in the body (“analyte”) out through the biologicalmembrane or tissue so that the analyte molecule may be collected on theoutside of the body.

As used herein, the term “bioactive agent,” “permeant,” “drug,” or.“pharmacologically active agent” or “deliverable substance” or any othersimilar term means any chemical or biological material or compoundsuitable for delivery by the methods previously known in the art and/orby the methods taught in the present invention, that induces a desiredeffect, such as a biological or pharmacological effect, which mayinclude but is not limited to (1) having a prophylactic effect on theorganism and preventing an undesired biological effect such aspreventing an infection, (2) alleviating a condition caused by adisease, for example, alleviating pain or inflammation caused as aresult of disease, (3) either alleviating, reducing, or completelyeliminating the disease from the organism, and/or (4) the placementwithin the viable tissue layers of the organism of a compound orformulation which can react, optionally in a reversible manner, tochanges in the concentration of a particular analyte and in so doingcause a detectable shift in this compound or formulation's measurableresponse to the application of energy to this area which may beelectromagnetic, mechanical or acoustic. The effect may be local, suchas providing for a local anesthetic effect, or it may be systemic. Thisinvention is not drawn to novel permeants or to new classes of activeagents other than by virtue of the microporation technique, althoughsubstances not typically being used for transdermal, transmucosal,transmembrane or transbuccal delivery may now be useable. Rather it isdirected to the mode of delivery of bioactive agents or permeants thatexist in the art or that may later be established as active agents andthat are suitable for delivery by the present invention.

Such substances include broad classes of compounds normally deliveredinto the organism, including through body surfaces and membranes,including skin as well as by injection, including needle, hydraulic, orhypervelocity methods. In general, this includes but is not limited to:Polypeptides, including proteins and peptides (e.g., insulin); releasingfactors, including Luteinizing Hormone Releasing Hormone (LHRH);carbohydrates (e.g., heparin); nucleic acids; vaccines; andpharmacologically active agents such as antiinfectives such asantibiotics and antiviral agents; analgesics and analgesic combinations;anorexics; antihelminthics; antiarthritics; antiasthmatic agents;anticonvulsants; antidepressants; antidiabetic agents; antidiarrheals;antihistamines; antiinflammatory agents; antimigraine preparations;antinauseants; antineoplastics; antiparkinsonism drugs; antipruritics;antipsychotics; antipyretics; antispasmodics; anticholinergics;sympathomimetics; xanthine derivatives; cardiovascular preparationsincluding potassium and calcium channel blockers, beta-blockers,alpha-blockers, and antiarrhythmics; antihypertensives; diuretics andantidiuretics; vasodilators including general coronary, peripheral andcerebral; central nervous system stimulants; vasoconstrictors; cough andcold preparations, including decongestants; hormones such as estradiol,testosterone, progesterone and other steroids and derivatives andanalogs, including corticosteroids; hypnotics; immunosuppressives;muscle relaxants; parasympatholytics; psychostimulants; sedatives; andtranquilizers. By the method of the present invention, both ionized andnonionized permeants may be delivered, as can permeants of any molecularweight including substances with molecular weights ranging from lessthan 50 Daltons to greater than 1,000,000 Daltons.

As used herein, an “effective” amount of a permeant means a sufficientamount of a compound to provide the desired local or systemic effect andperformance at a reasonable benefit/risk ratio attending any treatment.An “effective” amount of an enhancer as used herein means an amountselected so as to provide the desired increase in tissue permeabilityand the desired depth of penetration, rate of administration, and amountof permeant delivered.

As used herein, “carriers” or “vehicles” refer to carrier materialswithout significant pharmacological activity at the quantities used thatare suitable for administration with other permeants, and include anysuch materials known in the art, e.g., any liquid, gel, solvent, liquiddiluent, solubilizer, microspheres, liposomes, microparticles, lipidcomplexes, or the like, that is sufficiently nontoxic at the quantitiesemployed and does not interact with the drug to be administered in adeleterious manner. Examples of suitable carriers for use herein includewater, buffers, mineral oil, silicone, inorganic or organic gels,aqueous emulsions, liquid sugars, lipids, microparticles, waxes,petroleum jelly, and a variety of other oils and polymeric materials.

As used herein, a “biological membrane” means a tissue material presentwithin a living organism that separates one area of the organism fromanother and, in many instances, that separates the organism from itsouter environment. Skin and mucous and buccal membranes are thusincluded as well as the outer layers of a plant. Also, the walls of acell or a blood vessel would be included within this definition.

As used herein, “mucous membrane” or “mucosa” refers to the epitheliallinings of the mouth, nasopharynx, throat, respiratory tract, urogenitaltract, anus, eye, gut and all other surfaces accessible via anendoscopic device such as the bladder, Colon, lung, blood vessels, heartand the like.

As used herein, the “buccal membrane” includes the mucous membrane ofthe mouth.

As used herein, “outer layer” and “connective-tissue layer” are parts ofthe biological membrane and have the following meanings. “Outer layer”means all or part of the epidermis of the skin, or the epithelial liningof the mucous membrane or the outer layer of a plant. The mostsuperficial portion of the animal epidermis is the stratum corneum, asis well known in the art. The deeper portion of the epidermis is called,for simplicity, the “viable cell layer” hereinafter. Beneath the outerlayer is the “connective tissue layer.” The connective tissue layermeans the dermis in the skin or the lamina propria in the mucousmembrane or other underlying tissues in plants or animals.

As used herein, “organism” or “individual” or “subject” or “body” refersto any of a human, animal, or plant to which the present invention maybe applied.

As used herein, “analyte” means any chemical or biological material orcompound suitable for passage through a biological membrane by thetechnology taught in this present invention, or by technology previouslyknown in the art, of which an individual might want to know theconcentration or activity inside the body. Glucose is a specific exampleof an analyte because it is a sugar suitable for passage through theskin, and individuals, for example those having diabetes, might want toknow their blood glucose levels. Other examples of analytes include, butare not limited to, such compounds as sodium, potassium, bilirubin,urea, ammonia, calcium, lead, iron, lithium, salicylates, antibodies,hormones, or an exogenously delivered substance and the like.

As used herein, “into” or “in” a biological membrane or layer thereofincludes penetration in or through only one or more layers (e.g., all orpart of the stratum corneum or the entire outer layer of the skin orportion thereof).

As used herein, “through” a biological membrane or layer thereof meansthrough the entire depth of the biological membrane or layer thereof.

As used herein, “transdermal flux rate” is the rate of passage of anyanalyte out through the skin of a subject or the rate of passage of anybioactive agent, drug, pharmacologically active agent, dye, particle orpigment in and through the skin separating the organism from its outerenvironment. “Transmucosal flux rate” and “transbuccal flux rate” referto such passage through mucosa and buccal membranes and “transmembraneflux rate” refers to such passage through any biological membrane.

As used herein, “transdermal,” “transmucosal,” “transbuccal” and“transmembrane” may be used interchangeably as appropriate within thecontext of their use.

As used herein, the terms “intensity amplitude,” “intensity,” and“amplitude” are used synonymously and refer to the amount of energybeing produced by a sonic, thermal, mechanical or electromagnetic energysystem.

As used herein, “frequency modulation” or “sweep” means a continuous,graded or stepped variation in the frequency of a sonic, thermal,mechanical or electromagnetic energy in a given time period. A frequencymodulation is a graded or stepped variation in frequency in a given timeperiod, for example 5.4-5.76 MHz in 1 sec., or 5-10 MHz in 0.1 sec., or10-5 MHz in 0.1 sec., or any other frequency range or time period thatis appropriate to a specific application. A complex modulation caninclude varying both the frequency and intensity simultaneously. Forexample, FIGS. 4A and 4B of U.S. Pat. No. 5,458,140 could, respectively,represent amplitude and frequency modulations being appliedsimultaneously to a single sonic energy transducer.

As used herein, “amplitude modulation” means a continuous, graded orstepped variation in the amplitude or intensity of a sonic, thermal,mechanical or electromagnetic energy in a given time period.

As used herein “phase modulation” means the timing of a sonic, thermal,mechanical or electromagnetic energy or signal has been changed relativeto its initial state. An example is shown in FIG. 4C of U.S. Pat. No.5,458,140. The frequency and amplitude of the signal can remain thesame. A phase modulation can be implemented with a variable delay suchas to selectively retard or advance the signal temporarily in referenceto its previous state, or to another signal.

As used herein “signal,” or “energy” may be used synonymously. Thesonic, thermal, mechanical or electromagnetic energy, in its variousapplications such as with frequency, intensity or phase modulation, orcombinations thereof and the use of chemical enhancers combined withsonic, thermal, mechanical or electromagnetic energy, as describedherein, can vary over a frequency range of between about 0.01 Hz to 1000MHz, with a range of between about 0.1 Hz and 30 MHz being preferred.

As used herein, “non-invasive” means not requiring the entry of aneedle, catheter, or other invasive instrument into a part of thesubject including the skin or a mucous membrane.

As used herein, “minimally invasive and “non-invasive” are synonymous.

As used herein, “microparticles” or “microspheres” or “nanoparticles” or“nanospheres” or “ liposomes” or “lipid complexes” may be usedinterchangeably.

Means for Poration of the Biological Membrane

The formation of a micropore in the biological membrane can beaccomplished by various state of the art means as well as certain meansdisclosed herein that are improvements thereof. While the followingtechniques and examples are made with respect to porating the biologicalmembrane, it should be understood that the improvements described hereinalso apply to porating the mucous or buccal membrane or the outer layersof a plant.

The use of laser ablation as described by Jacques et al. in U.S. Pat.No. 4,775,361 and by Lane et al., supra, certainly provide one means forablating the stratum corneum using an excimer laser. At 193 nmwavelength, and 14 ns pulse width, it was found that about 0.24 to 2.8μnm of stratum corneum could be removed by each laser pulse at radiantexposure of between about 70 and 480 mJ/cm². As the pulse energyincreases, more tissue is removed from the stratum corneum and fewerpulses are required for complete poration of this layer. The lowerthreshold of radiant exposure that must be absorbed by the stratumcorneum within the limit of the thermal relaxation time to causesuitable micro-explosions that result in tissue ablation is about 70mJ/cm² within a 50 millisecond (ms) time. In other words, a total of 70mJ/cm² must be delivered within a 50 ms window. This can be done in asingle pulse of 70 mJ/cm² or in 10 pulses of 7 mJ/cm², or with acontinuous illumination of 1.4 watts/cm² during the 50 ms time. Theupper limit of radiant exposure is that which will ablate the stratumcorneum without damage to underlying tissue and can be empiricallydetermined from the light source, wavelength of light, and othervariables that are within the experience and knowledge of one skilled inthis art.

By “delivery”, in the context of the application of energy, is meantthat the stated amount of energy is absorbed by the tissue to beablated. At the excimer laser wavelength of 193 nm, essentially 100%absorption occurs within the first 1 or 2 μm of stratum corneum tissue.Assuming the stratum corneum is about 20 μm thick, at longerwavelengths, such as 670 nm, only about 5% of incident light is absorbedwithin the 20 μm layer. This means that about 95% of the high power beampasses into the tissues underlying the stratum corneum where it willlikely cause significant damage. In the context of delivery of abioactive agent, the term means providing the bioactive agent to thedesired location.

The ideal is to use only as much power as is necessary to perforate thebiological membrane or other selected skin, mucosal, or tissue layerswithout causing bleeding, thermal, or other unacceptable damage tounderlying and adjacent tissues from which analytes are to be extractedor permeants delivered.

It would be beneficial to use sources of energy more economical thanenergy from excimer lasers. Excimer lasers, which emit light atwavelengths in the far UV region, are much more expensive to operate andmaintain than, for example, diode lasers that emit light at wavelengthsin visible and IR regions (600 to 1800 nm). However, at the longerwavelengths, the biological membrane becomes increasingly moretransparent and absorption occurs primarily in the underlying tissues.

The present invention facilitates a rapid and minimally traumatic methodof eliminating the barrier function of the biological membrane tofacilitate the transmembrane transport of substances into the body whenapplied topically or to access the analytes within the body foranalysis. The method utilizes a procedure which begins with the contactapplication of a small area heat source to the targeted area of thebiological membrane.

The heat source must have several important properties, as will now bedescribed. First, the heat source must be sized such that contact withthe biological membrane is confined to a small area, typically about 1to 1000 μm in diameter. Second, it must have the capability to modulatethe temperature of the biological membrane at the contact point fromambient surface temperature to greater than the vaporization point of asufficient amount of the components within the biological membrane andthen return to approximately ambient temperature with cycle times tominimize collateral damage to viable tissues and trauma to the subject.This modulation can be created electronically, mechanically, orchemically.

Additionally, for selected applications, an inherent depth limitingfeature of the microporation process can be facilitated if the heatsource has both a small enough thermal mass and limited energy source toelevate its temperature such that when it is placed in contact withtissues with more than 30% water content, the thermal dispersion inthese tissues is sufficient to limit the maximum temperature of the heatsource to less than 100 C. This feature effectively stops the thermalvaporization process once the heat probe had penetrated through thestratum corneum into or through the lower layers of the epidermis.

However, if one utilizes a heat probe which can continue to deliversufficient energy into or through the hydrated viable tissue layersbeneath the outer layer of the biological membrane, the poration processcan continue into the body to a selected depth, penetrating throughdeeper layers including, e.g., in the case of the skin, through theepidermis, the dermis, and into the subcutaneous layers below ifdesired. The concern when a system is designed to create a microporeextending some distance into or through the viable tissues beneath thestratum corneum, mucosal or buccal membranes is principally how tominimize damage to the adjacent tissue and the sensation to the subjectduring the poration process. Experimentally, we have shown that if theheat probe used is a solid, electrically or optically heated element,with the active heated probe tip physically defined to be no more than afew hundred microns across and protruding up to a few millimeters fromthe supporting base, that a single pulse, or multiple pulses of currentcan deliver enough thermal energy into or through the tissue to allowthe ablation to penetrate as deep as the physical design allows, forexample, until the support base acts as a component to limit the extentof the penetration into or through the tissue, essentially restrictingthe depth to which the heat probe can penetrate into a micropore tocontact fresh, unporated tissue. If the electrical and thermalproperties of said heat probe, when it is in contact with the tissues,allow the energy pulse to modulate the temperature of said probe rapidlyenough, this type of deep tissue poration can be accomplished withessentially no pain to the subject. Experiments have shown that if therequired amount of thermal energy is delivered to the probe within lessthan roughly 20 milliseconds, that the procedure is painless.Conversely, if the energy pulse must be extended beyond roughly 20milliseconds, the sensation to the subject increases rapidly andnon-linearly as the pulse width is extended.

An electrically heated probe design which supports this type of selecteddepth poration can be built by bending a 50 to 150 micron diametertungsten wire into a sharp kink, forming a close to 180 degree bend witha minimal internal radius at this point. This miniature ‘V’ shaped pieceof wire can then be mounted such that the point of the ‘V’ extends somedistance out from a support piece which has copper electrodes depositedupon it. The distance to which the wire extends out from the supportwill define the maximum penetration distance into or through the tissuewhen the wire is heated. Each leg of the tungsten ‘V’ will be attachedto one of the electrodes on the support carrier which in turn can beconnected to the current pulsing circuit. When the current is deliveredto the wire in an appropriately controlled fashion, the wire willrapidly heat up to the desired temperature to effect the thermalablation process in a single pulse or in multiple pulses of current. Bymonitoring the dynamic impedance of the probe and knowing thecoefficient of resistance versus temperature of the tungsten element,closed loop control of the temperature of the contact point can easilybe established. Also; by dynamically monitoring the impedance throughthe body from the contact point of the probe and a second electrodeplaced some distance away, the depth of the pore can be estimated basedon the different impedance properties of the tissue as one penetratesdeeper into the body.

An optically heated probe design which supports this type of selecteddepth poration can be built by taking an optical fiber and placing onone end a tip comprised of a solid cap or coating. A light source suchas a laser diode will be coupled into the other end of the fiber. Theside of tip facing the fiber must have a high enough absorptioncoefficient over the range of wavelengths emitted by the light sourcethat when the photons reach the end of the fiber and strike this face,some of them will be absorbed and subsequently cause the tip to heat up.The specific design of this tip, fiber and source assembly may varywidely, however fibers with gross diameters of 50 to 1000 microns acrossare common place items today and sources emitting up to thousands ofwatts of optical energy are similarly common place. The tip forming theactual heat probe can be fabricated from a high melting point material,such as tungsten and attached to the fiber by machining it to allow theinsertion of the fiber into a cylindrical bore at the fiber end. If thedistal end of the tip has been fabricated to limit the thermal diffusionaway from this tip and back up the supporting cylinder attaching the tipto the fiber within the time frame of the optical pulse widths used, thephotons incident upon this tip will elevate the temperature rapidly onboth the fiber side and the contact side which is placed against thetissues surface. The positioning of the fiber/tip assembly onto thetissue surface, can be accomplished with a simple mechanism designed tohold the tip against the surface under some spring tension such that asthe tissue beneath it is ablated, the tip itself will advance into thetissue. This allows the thermal ablation process to continue into orthrough the tissue as far as one desires. An additional feature of thisoptically heated probe design is that by monitoring the black bodyradiated energy from the heated tip that is collected by the fiber, avery simple closed loop control of the tip temperature can be effected.Also, as described earlier, by dynamically monitoring the impedancethrough the body from the contact point of the probe and a secondelectrode placed some distance away, the depth of the pore can bedetermined based on the different impedance properties of the tissue asone penetrates deeper into the body. The relationship between pulsewidth and sensation for this design is essentially the same as for theelectrically heated probe described earlier.

Impedance can be used to determine the depth of a pore made by anymeans. It can be used as an input to a control system for making poresof selected depth. The impedance measured may be the complex impedancemeasured at a frequency selected to highlight the impedance propertiesof the selected tissues in a selected organism.

An additional feature of this invention is the large increase inefficiency which can be gained by combining the poration of the outerlayers of the biological membrane with other permeation enhancementtechniques which can now be optimized to function on the variousbarriers to effective delivery of the desired compound into or throughthe internal spaces it needs to go to be bio-effective. In particular,if one is delivering a DNA compound either naked, fragmented,encapsulated or coupled to another agent, it is often desired to get theDNA into the living cells without killing the cell to allow the desireduptake and subsequent performance of the therapy. It is well know in theart that electroporation, iontophoresis, and ultrasound can causeopenings to form, temporarily, in the cell membranes and other internaltissue membranes. By having breached the stratum corneum or mucosallayer or outer layer of a plant and if desired the epidermis and dermisor deeper into a plant, electroporation, iontophoresis, magnetic fields,and sonic energy can now be used with parameters that can be tailored toact selectively on these underlying tissue barriers. For example, forany electromagnetic or sonic energy enhancement means, the specificaction of the enhancement can be designed to focus on any part of thepore, e.g., on the bottom of the pore by the design of the focusingmeans employed such as the design of the electrodes, sonic and magneticfield forming devices and the like. Alternatively, the enhancer can befocused more generally on the entire pore or the area surrounding thepore. In the case of electroporation, where pulses exceeding 50 to 150volts are routinely used to electroporate the stratum corneum or mucosallayer, in the environment we present, pulses of only a few volts can besufficient to electroporate the cell, capillary or other membraneswithin the targeted tissue. This is principally due to the dramaticreduction in the number of insulating layers present between theelectrodes once the outer surface of the biological membrane has beenopened. Similarly, iontophoresis can be shown to be effective tomodulate the flux of a fluid media containing the DNA through themicropores with very small amounts of current due to the dramaticreduction in the physical impedance to fluid flow through these poratedlayers.

Whereas ultrasound has previously been used to accelerate the permeationof the stratum corneum or mucosal layer, by eliminating this barrier viathe micropores, we have created the opportunity to utilize sonic energyto permeabilize the cell, capillary or other structures within thetargeted tissue. As in the cases of electroporation and iontophoresis,we have demonstrated that the sonic energy levels needed to effect anotable improvement in the trans-membrane flux of a substance are muchlower than when stratum corneum or mucosal layers are left intact. Themode of operation of all of these active methods, electroporation,iontophoresis, magnetic fields, mechanical forces or ultrasound, whenapplied solely or in combination, after the poration of biologicalmembrane has been effected is most similar to the parameters typicallyused in in vitro applications where single cell membranes are beingopened up for the delivery of a substance.

With the heat source placed in contact with the surface of thebiological membrane, it is cycled through a series of one or moremodulations of temperature from an initial point of ambient temperatureto a peak temperature in excess of 123° C. and back to ambient surfacetemperature. To minimize or eliminate the animal's sensory perception ofthe microporation process, these pulses are limited in duration, and theinterpulse spacing is long enough to allow cooling of the viable tissuelayers in the biological membrane, and most particularly the innervatedtissues, to achieve a mean temperature within the innervated tissues ofless than about 45 C. These parameters are based on the thermal timeconstants of the human skin's viable epidermal tissues (roughly 30-80ms) located between the heat probe and the innervated tissue in theunderlying dermis. The result of this application of pulsed thermalenergy is that enough energy is conducted into or through the stratumcorneum within the tiny target spot that the local temperature of thisvolume of tissue is elevated sufficiently higher than the vaporizationpoint of the tissue-bound water content in the stratum corneum. As thetemperature increases above 100 C, the water content of the stratumcorneum (typically 5% to 15%) within this localized spot, is induced tovaporize and expand very rapidly, causing a vapor-driven removal ofthose corneocytes in the stratum corneum located in proximity to thisvaporization event. U.S. Pat. No. 4,775,361 teaches that a stratumcorneum temperature of 123° C. represents a threshold at which this typeof flash vaporization occurs. As subsequent pulses of thermal energy areapplied, additional layers of the stratum corneum are removed until amicropore is formed through the stratum corneum down to the next layerof the epidermis, the stratum lucidum. By limiting the duration of theheat pulse to less than one thermal time constant of the epidermis andallowing any heat energy conducted into or through the epidermis todissipate for a sufficiently long enough time, the elevation intemperature of the viable layers of the epidermis is minimal. Thisallows the entire microporation process to take place without anysensation to the subject and no damage to the underlying and surroundingtissues. If the heat probe can achieve temperatures greater than 300degrees C. some of the poration may be due to the direct thermaldecomposition of the tissue.

The present invention comprises a method for painlessly, or with littlesensation, creating microscopic holes, i.e. micropores, from about 1 to1000 μm across, in a biological membrane of an organism. The key tosuccessfully implementing this method is the creation of an appropriatethermal energy source, or heat probe, which is held in contact with thebiological membrane. The principle technical challenge in fabricating anappropriate heat probe is designing a device that has the desiredcontact with the biological membrane and that can be thermally modulatedat a sufficiently high frequency.

It is possible to fabricate an appropriate heat probe by contacting thebiological membrane with a suitable light-absorbing compound, such as adye or stain, or any thin film or substance selected because of itsability to absorb light at the wavelength emitted by a selected lightsource. In this instance, the selected light source may be a laser diodeemitting at a wavelength which would not normally be absorbed by thebiological membrane. By focusing the light source to a small spot on thesurface of the topical layer of the dye, stain, thin film or substancethe targeted area can be temperature modulated by varying the intensityof the light flux focused on it. It is possible to utilize the energyfrom laser sources emitting at a longer wavelength than an excimer laserby first topically applying to the stratum corneum a suitablelight-absorbing compound, such as a dye, stain, thin film or substanceselected because of its ability to absorb light at the wavelengthemitted by the laser source. The same concept can be applied at anywavelength and one must only choose an appropriate dye or stain andoptical wavelength. One need only look to any reference manual to findwhich suitable dyes and wavelength of the maximum absorbance of thatdye. One such reference is Green, The Sigma-Aldrich Handbook of Stains.Dyes and Indicators, Aldrich Chemical Company, Inc. Milwaukee, Wis.(1991). For example, copper phthalocyanine (Pigment Blue 15; CPC)absorbs at about 800 nm; copper phthalocyanine tetrasulfonic acid (AcidBlue 249) absorbs at about 610 nm; and Indocyanine Green absorbs atabout 775 nm; and Cryptocyanine absorbs at about 703 nm. CPC isparticularly well suited for this embodiment for the following reasons:it is a very stable and inert compound, already approved by the FDA foruse as a dye in implantable sutures; it absorbs very strongly atwavelengths from 750 nm to 950 nm, which coincide well with numerous lowcost, solid state emitters such as laser diodes and LEDs, and inaddition, this area of optical bandwidth is similarly not absorbeddirectly by the skin tissues in any significant amount; CPC has a veryhigh vaporization point (>550 C in a vacuum) and goes directly from asolid phase to a vapor phase with no liquid phase; CPC has a relativelylow thermal diffusivity constant, allowing the light energy focused onit to selectively heat only that area directly in the focal point withvery little lateral spreading of the ‘hot-spot’ into the surrounding CPCthereby assisting in the spatial definition of the contact heat-probe.

The purpose of this disclosure is not to make an exhaustive listing ofsuitable dyes, stains, films or substances because such may be easilyascertained by one skilled in the art from data readily available.

The same is true for any desired particular pulsed light source. Forexample, this method may be implemented with a mechanically shuttered,focused incandescent lamp as the pulsed light source. Various catalogsand sales literature show numerous lasers operating in the near UV,visible and near IR range. Representative lasers are Hammamatsu PhotonicSystems Model PLP-02 which operates at a power, output of 2×10⁻⁸ J, at awavelength of 415 nm; Hammamatsu Photonic Systems Model PLP-05 whichoperates at a power output of 15 J, at a wavelength of 685 nm; SDL,Inc., SDL-3250 Series pulsed laser which operates at a power output of2×10⁶ J at a wavelength of about 800-810 nm; SDL, Inc., Model SDL-8630which operates at a power output of 500 mW at a wavelength of about 670nm; Uniphase Laser Model AR-081-15000 which operates at a power outputof 15,000 mW at a wavelength of 790-830 nm; Toshiba America ElectronicModel TOLD9150 which operates at a power output of 30 mW at a wavelengthof 690 nm; and LiCONIX, Model Diolite 800-50 which operates at a power50 mW at a wavelength of 780 nm.

For purposes of the present invention a pulsed laser light source canemit radiation over a wide range of wavelengths ranging from betweenabout 100 nm to 12,000 nm. Excimer lasers typically will emit over arange of between about 100 to 400 nm. Commercial excimer lasers arecurrently available with wavelengths in the range of about 193 nm to 350nm. Preferably a laser diode will have an emission range of betweenabout 380 to 1550 nm. A frequency doubled laser diode will have anemission range of between about 190 and 775 nm. Longer wavelengthsranging from between about 1300 and 3000 nm may be utilized using alaser diode pumped optical parametric oscillator. It is expected, giventhe amount of research taking place on laser technology, that theseranges will expand with time.

Delivered or absorbed energy need not be obtained from a laser as anysource of light, whether it is from a laser, a short arc lamp such as axenon flashlamp, an incandescent lamp, a light-emitting diode (LED), thesun, or any other source may be used. Thus, the particular instrumentused for delivering electromagnetic radiation is less important than thewavelength and energy associated therewith. Any suitable instrumentcapable of delivering the necessary energy at suitable wavelengths, i.e.in the range of about 100 nm to about 12,000 nm, can be consideredwithin the scope of the invention. The essential feature is that theenergy must be absorbed by the light-absorbing compound to causelocalized heating thereof, followed by conduction of sufficient heat tothe tissue to be ablated within the time frame allowed.

In one illustrative embodiment, the heat probe itself is formed from athin layer, preferably about 5 to 1000 μm thick, of a solid,non-biologically active substance placed in contact with a selected areaof an individual's skin that is large enough to cover the site where amicropore is to be created. The specific formulation of the chemicalcompound is chosen such that it exhibits high absorption over thespectral range of a light source selected for providing energy to thelight-absorbing compound. The probe can be, for example, a sheet of asolid compound, a film treated or coated with or containing a suitablelight absorbing compound, or a direct application of the light-absorbingcompound to the skin as a precipitate or as a suspension in a carrier.Regardless of the configuration of the light-absorbing heat probe, itmust exhibit a low enough lateral thermal diffusion coefficient suchthat any local elevations of temperature will remain sufficientlyspatially defined and the dominant mode of heat loss will preferably bevia direct conduction into biological membrane through the point ofcontact between the skin and the probe.

The required temperature modulation of the probe can be achieved byfocusing a light source onto the probe layer and modulating theintensity of this light source. If the energy absorbed within theilluminated area is sufficiently high, it will cause the probe layerheat up. The amount of energy delivered, and subsequently both the rateof heating and peak temperature of the probe layer at the focal point,can be easily modulated by varying the pulse width and peak power of thelight source. In this embodiment, it is only the small volume of probelayer heated up by the focused, incident optical energy that forms theheat probe, additional material of this probe layer which may have beenapplied over a larger area then the actual poration site is incidental.By using a solid phase light-absorbing compound with a relatively highmelting point, such as copper phthalocyanine (CPC), which remains in itssolid phase up to a temperature of greater than 550 C, the heat probecan be quickly brought up to a temperature of several hundred degreesC., and still remain in contact with the biological membrane, allowingthis thermal energy, to be conducted into or through the stratumcorneum. In addition, this embodiment comprises choosing a light sourcewith an emission spectrum where very little energy would normally beabsorbed in the tissues of the biological membrane.

Once the targeted area has the light-absorbing probe layer placed incontact to it, the heat probe is formed when the light source isactivated with the focal waist of the beam positioned to be coincidentwith the surface of the treated area The energy density of light at thefocal waist and the amount of absorption taking place within thelight-absorbing compound are set to be sufficient to bring thetemperature of the light-absorbing compound, within the area of thesmall spot defined by the focus of the light source, to greater than123° C. within a few milliseconds. As the temperature of the heat proberises, conduction into or through the biological membrane deliversenergy into or through these tissues, elevating the local temperature ofthe biological membrane. When enough energy has been delivered into orthrough this small area of biological membrane to cause the localtemperature to be elevated above the boiling point of some of the waterand other vaporizable components contained in these tissues, a flashvaporization of this material takes place, removing some portion of thebiological membrane at this location and forming a micropore.

By turning the light source on and off, the temperature of the heatprobe can be rapidly modulated and the selective ablation of thesetissues can be achieved, allowing a very precisely dimensioned hole tobe created, which can selectively penetrate only through the first 10 to30 microns of the biological membrane, or can be made deeper.

An additional feature of this embodiment is that by choosing a lightsource that would normally have very little energy absorbed by thebiological membrane or underlying tissues, and by designing the focusingand delivery optics to have a sufficiently high numerical aperture, thesmall amount of delivered light that does not happen to get absorbed inthe heat probe itself, quickly diverges as it penetrates deep into thebody. Since there is very little absorption at the deliveredwavelengths, essentially no energy is delivered to the biologicalmembrane directly from the light source. This three dimensional dilutionof coupled energy in the tissues due to beam divergence and the lowlevel of absorption in the untreated tissue results in a completelybenign interaction between the light beam and the tissues, with nodamage being done thereby.

In one preferred embodiment of the invention, a laser diode is used asthe light source with an emission wavelength of 800±30 nm. A heat-probecan be formed by topical application of a transparent adhesive tape thathas been treated on the adhesive side with a 0.5 cm spot formed from adeposit of finely ground copper phthalocyanine (CPC). The CPC exhibitsextremely high absorption coefficients in the 800 nm spectral range,typically absorbing more than 95% of the radiant energy from a laserdiode.

FIG. 1 shows a system 10 for delivering light from such a laser diode toa selected area of an individual's biological membrane and formonitoring the progress of the poration process. The system comprises alaser diode 14 coupled to a controller 18, which controls the intensity,duration, and spacing of the light pulses. The laser diode emits a beam22 that is directed to a collection lens or lenses 26, which focuses thebeam onto a mirror 30. The beam is then reflected by the mirror to anobjective lens or lenses 34, which focuses the beam at a preselectedpoint 38. This preselected point corresponds with the plane of an xyzstage 42 and the objective hole 46 thereof, such that a selected area ofan individual's biological membrane can be irradiated. The xyz stage isconnected to the controller such that the position of the xyz stage canbe controlled. The system also comprises a monitoring system comprisinga CCD camera 50 coupled to a monitor 54. The CCD camera is confocallyaligned with the objective lens such that the progress of the porationprocess can be monitored visually on the monitor.

In another illustrative embodiment of the invention, a system of sensingphotodiodes and collection optics that have been confocally aligned withthe ablation light source is provided. FIG. 2 shows a sensor system 60for use in this embodiment. The system comprises a light source 64 foremitting a beam of light 68, which is directed through a delivery opticssystem 72 that focuses the beam at a preselected point 76, such as thesurface of an individual's skin 80. A portion of the light contactingthe skin is reflected, and other light is emitted from the irradiatedarea. A portion of this reflected and emitted light passes through afilter 84 and then through a collection optics system 88, which focusesthe light on a phototodiode 92. A controller 96 is coupled to both thelaser diode and the photodiode for, respectively, controlling the outputof the laser diode and detecting the light that reaches the photodiode.Only selected portions of the spectrum emitted from the skin passthrough the filter. By analyzing the shifts in the reflected and emittedlight from the targeted area, the system has the ability to detect whenthe stratum corneum has been breached, and this feedback is then used tocontrol the light source, deactivating the pulses of light when themicroporation of the stratum corneum is achieved. By employing this typeof active closed loop feedback system, a self regulating, universallyapplicable device is obtained that produces uniformly dimensionedmicropores in the stratum corneum, with minimal power requirements,regardless of variations from one individual to the next.

In another illustrative embodiment, a cooling device is incorporatedinto the system interface to the skin. FIG. 3A shows an illustrativeschematic representation thereof. In this system 100, a light source 104(coupled to a controller 106) emits a beam of light 108, which passesthrough and is focused by a delivery optics system 112. The beam isfocused by the delivery optics system to a preselected point 116, suchas a selected area of an individual's skin 120. A cooling device 124,such as a Peltier device or other means of chilling, contacts the skinto cool the surface thereof. In a preferred embodiment of the coolingdevice 124 (FIG. 3B), there is a central hole 128 through which the beamof focused light passes to contact the skin. Referring again to FIG. 3A,a heat sink 132 is also preferably placed in contact with the coolingdevice. By providing a cooling device with a small hole in its centercoincident with the focus of the light, the tissues in the general areawhere the poration is to be created may be cooled to 5° C. to 10° C.This cooling allows a greater safety margin for the system to operate inthat the potential sensations to the user and the possibility of anycollateral damage to the epidermis directly below the poration site arereduced significantly from non-cooled embodiment. Moreover, formonitoring applications, cooling minimizes evaporation of interstitialfluid and can also provide advantageous physical properties, such asdecreased surface tension of such interstitial fluid. Still further,cooling the tissue is known to cause a localized increase in blood flowin such cooled tissue, thus promoting diffusion of analytes from theblood into the interstitial fluid and promoting diffusion of deliveredpermeants away from the pore site or into the tissue underlying thepore.

The method can also be applied for other micro-surgery techniqueswherein the light-absorbing compound/heat-probe is applied to the areato be ablated and then the light source is used to selectively modulatethe temperature of the probe at the selected target site, affecting thetissues via the vaporization-ablation process produced.

A further feature of the invention is to use the light source to helpseal the micropore after its usefulness has passed. Specifically, in thecase of monitoring for an internal analyte, a micropore is created andsome amount of interstitial fluid is extracted through this opening.After a sufficient amount of interstitial fluid had been collected, thelight source is reactivated at a reduced power level to facilitate rapidclotting or coagulation of the interstitial fluid within the micropore.By forcing the coagulation or clotting of the fluid in the pore, thisopening in the body is effectively sealed, thus reducing the risk ofinfection. Also, the use of the light source itself for both theformation of the micropore and the sealing thereof is an inherentlysterile procedure, with no physical penetration into the body by anydevice or apparatus. Further, the thermal shock induced by the lightenergy kills any microbes that may happen to be present at the ablationsite.

This concept of optical sterilization can be extended to include anadditional step in the process wherein the light source is first appliedin an unfocused manner, covering the target area with an illuminatedarea that extends 100 μm or more beyond the actual size of the microporeto be produced. By selecting the area over which the unfocused beam isto be applied, the flux density can be correspondingly reduced to alevel well below the ablation threshold but high enough to effectivelysterilize the surface of the skin. After a sufficiently long exposure ofthe larger area, either in one continuous step or in a series of pulses,to the sterilizing beam, the system is then configured into the sharplyfocused ablation mode and the optical microporation process begins.

Another illustrative embodiment of the invention is to create therequired heat probe from a solid element, such as a small diameter wire.As in the previously described embodiment, the contacting surface of theheat probe must be able to have its temperature modulated from ambientbiological membrane temperatures to temperatures greater than 123° C.,within the required time allowed of, preferably, between about 1microsecond to 50 milliseconds at the high temperature (on-time) and atleast about 1 to 50 ms at the low temperature (off-time). In particular,being able to modulate the temperature up to greater than 150° C. for an“on” time of around 5 ms and an off time of 50 ms produces veryeffective thermal ablation with little or no sensation to theindividual.

Several methods for modulating the temperatures of the solid elementheat probe contact area may be successfully implemented. For example, ashort length of wire may be brought up to the desired high temperatureby an external heating element such as an ohmic heating element used inthe tip of a soldering iron. FIG. 4 shows an ohmic heating device 140with a mechanical actuator. The ohmic heating device comprises an ohmicheat source 144 coupled to a solid element heat probe 148. The ohmicheat source is also coupled through an insulating mount 152 to amechanical modulation device 156, such as a solenoid. In thisconfiguration, a steady state condition can be reached wherein the tipof the solid element probe will stabilize at some equilibriumtemperature defined by the physical parameters of the structure, i.e.,the temperature of the ohmic heat source, the length and diameter of thesolid element, the temperature of the air surrounding the solid element,and the material of which the solid element is comprised. Once thedesired temperature is achieved, the modulation of the temperature ofthe selected area of an organism's biological membrane 160 is effecteddirectly via the mechanical modulation device to alternatively place thehot tip of the wire in contact with the biological membrane for,preferably, a 5 ms on-time and then withdraw it into the air for,preferably, a 50 ms off-time.

Another illustrative example (FIG. 5), shows a device 170 comprising acurrent source 174 coupled to a controller 178. The current source iscoupled to a current loop 182 comprising a solid element 186 formed intoa structure such that it presents a high resistance point. Preferably,the solid element is held on a mount 190, and an insulator 194 separatesdifferent parts of the current loop. The desired modulation oftemperature is then achieved by merely modulating the current throughthe solid element. If the thermal mass of the solid element isappropriately sized and the heat sinking provided by the electrodesconnecting it to the current source is sufficient, the warm-up andcool-down times of the solid element can be achieved in a fewmilliseconds. Contacting the solid element with a selected area ofbiological membrane 198 heats the biological membrane to achieve theselected ablation.

In FIG. 6 there is shown still another illustrative example of poratingthe biological membrane with a solid element heat probe. In this system200, the solid element 204 can be positioned within a modulatablealternating magnetic field formed by a coil of wire 208, the excitationcoil. By energizing the alternating current in the excitation coil bymeans of a controller 212 coupled thereto, eddy currents can be inducedin the solid element heat probe of sufficient intensity that it will beheated up directly via the internal ohmic losses. This is essentially aminiature version of an inductive heating system commonly used for heattreating the tips of tools or inducing out-gassing from the electrodesin vacuum or flash tubes. The advantage of the inductive heating methodis that the energy delivered into the solid element heat probe can beclosely controlled and modulated easily via the electronic control ofthe excitation coil with no direct electrical connection to the heatprobe itself. If the thermal mass of the solid element heat probe andthe thermal mass of the biological membrane in contact with the tip ofthe probe are known, controlling the inductive energy delivered canallow precise control of the temperature at the contact point 216 withthe biological membrane 220. Because the biological membrane tissue isessentially non-magnetic at the lower frequencies at which inductiveheating can be achieved, if appropriately selected frequencies are usedin the excitation coil, then this alternating electromagnetic field willhave no effect on the organism's tissues.

If a mechanically controlled contact modulation is employed, anadditional feature may be realized by incorporating a simple closed loopcontrol system wherein the electrical impedance between the probe tipand the subject's skin is monitored. In this manner, the probe can bebrought into contact with the subject's skin, indicated by the step-wisereduction in resistance once contact is made, and then held there forthe desired “on-time,” after which it can be withdrawn. Several types oflinear actuators are suitable for this form of closed loop control, suchas a voice-coil mechanism, a simple solenoid, a rotary system with a camor bell-crank, and the like. The advantage is that as the thermalablation progresses, the position of the thermal probe tip can besimilarly advanced into the biological membrane, always ensuring good acontact to facilitate the efficient transfer of the required thermalenergy. Also, for poration of skin, the change in the conductivityproperties of the stratum corneum and the epidermis can be used toprovide an elegant closed loop verification that the poration process iscomplete, i.e., when the resistance indicates that the epidermis hasbeen reached, it is time to stop the poration process. Similar changesin impedance can be used to control the depth of penetration to otherlayers as well.

FIG. 7 shows an illustrative example of such a closed loop impedancemonitor. In this system 230, there is an ohmic heat source 234 coupledto a wire heat probe 238. The heat source is mounted through aninsulating mount 242 on a mechanical modulator 246. A controller 250 iscoupled to the wire and to the skin 254, wherein the controller detectschanges in impedance in the selected area 258 of skin, and when apredetermined level is obtained the controller stops the porationprocess.

Along the same line as hydraulic poration means are microlancets adaptedto just penetrate the stratum corneum for purposes of administering apermeant, such as a drug, through the pore formed or to withdraw ananalyte through the pore for analysis. Such a device is considered to be“minimally invasive” as compared to devices and/or techniques which arenon-invasive. The use of micro-lancets that penetrate below the stratumcorneum for withdrawing blood are well known. Such devices arecommercially available from manufacturers such as Becton-Dickinson andLifescan and can be utilized in the present invention by controlling thedepth of penetration. As an example of a micro-lancet device forcollecting body fluids, reference is made to Erickson et al.,International Published PCT Application WO 95/10223 (published 20 Apr.1995). This application shows a device for penetration into or throughthe dermal layer of the skin, without penetration into subcutaneoustissues, to collect body fluids for monitoring, such as for bloodglucose levels.

Poration of a biological membrane can also be accomplished using sonicmeans. Sonic-poration is a variation of the optical means describedabove except that, instead of using a light source, a very tightlyfocused beam of sonic energy is delivered to the area of the stratumcorneum to be ablated. The same levels of energy are required, i.e. athreshold of 70 mJ/cm²/50 ms still must be absorbed. The same pulsedfocused ultrasonic transducers as described in parent applications Ser.Nos. 08/152,442 (now U.S. Pat. No. 5,458,140) and Ser. No. 08/152,174(now U.S. Pat. No. 5,445,611) can be utilized to deliver the requiredenergy densities for ablation as are used in the delivery of sonicenergy which is modulated in intensity, phase, or frequency or acombination of these parameters for the transdermal sampling of ananalyte or the transdermal delivery of drugs. This has the advantage ofallowing use of the same transducer to push a drug through the stratumcorneum or pull a body fluid to the surface for analysis to be used tofirst create a micropore.

Additionally, electroporation or short bursts or pulses of electricalcurrent can be delivered to the stratum corneum with sufficient energyto form micropores. Electroporation is known in the art for producingpores in biological membranes and electroporation instruments arecommercially available. Thus, a person of skill in this art can selectan instrument and conditions for use thereof without undueexperimentation according to the guidelines provided herein.

The micropores produced in the biological membrane by the methods of thepresent invention allow high flux rates of a variety of molecular weighttherapeutic compounds to be delivered transmembranely. In addition,these non-traumatic microscopic openings into the body allow access tovarious analytes within the body, which can be assayed to determinetheir internal concentrations.

Example 1

In this example, skin samples were prepared as follows. Epidermalmembrane was separated from human cadaver whole skin by theheat-separation method of Klingman and Christopher, 88 Arch. Dermatol.702 (1963), involving the exposure of the full thickness skin to atemperature of 60° C. for 60 seconds, after which time the stratumcorneum and part of the epidermis (epidermal membrane) were gentlypeeled from the dermis.

Example 2

Heat separated stratum corneum samples prepared according to theprocedure of. Example 1 were cut into 1 cm² sections. These smallsamples were than attached to a glass cover slide by placing them on theslide and applying an pressure sensitive adhesive backed disk with a 6mm hole in the center over the skin sample. The samples were then readyfor experimental testing. In some instances the skin samples werehydrated by allowing them to soak for several hours in a neutralbuffered phosphate solution or pure water.

As a test of these untreated skin samples, the outputs of severaldifferent infrared laser diodes, emitting at roughly 810, 905, 1480 and1550 nanometers were applied to the sample. The delivery optics weredesigned to produce a focal waist 25 μm across with a final objectivehave a numerical aperture of 0.4. The total power delivered to the focalpoint was measured to be between 50 and 200 milliwatts for the 810 and1480 nm laser diodes, which were capable of operating in a continuouswave (CW) fashion. The 905 and 1550 nm laser diodes were designed toproduce high peak power pulses roughly 10 to 200 nanoseconds, long atrepetition rates up to 5000 Hz. For the pulsed lasers the peak powerlevels were measured to be 45 watts at 905 nm and 3.5 watts at 1550 nm.

Under these operating conditions, there was no apparent effect on theskin samples from any of the lasers. The targeted area was illuminatedcontinuously for 60 seconds and then examined microscopically, revealingno visible effects. In addition, the sample was placed in a modifiedFranz cell, typically used to test transdermal delivery systems based onchemical permeation enhancers, and the conductivity from one side of themembrane to the other was measured both before and after the irradiationby the laser and showed no change. Based on these tests which were runon skin samples from four different donors, it was concluded that atthese wavelengths the coupling of the optical energy into or through theskin tissue was so small that no effects are detectable.

Example 3

To evaluate the potential sensation to a living subject when illuminatedwith optical energy under the conditions of Example 2, six volunteerswere used and the output of each laser source was applied to theirfingertips, forearms, and the backs of their hands. In the cases of the810, 905 and 1550 nm lasers, the subject was unable to sense when thelaser was turned on or off. In the case of the 1480 nm laser, there wasa some sensation during the illumination by the 1480 nm laser operatingat 70 mW CW, and a short while later a tiny blister was formed under theskin due to the absorption of the 1480 nm radiation by one of the waterabsorption bands. Apparently the amount of energy absorbed wassufficient to induce the formation of the blister, but was not enough tocause the ablative removal of the stratum corneum. Also, the absorptionof the 1480 nm light occurred predominantly in the deeper, fullyhydrated (85% to 90% water content) tissues of the epidermis and dermis,not the relatively dry (10% to 15% water content) tissue of the stratumcorneum.

Example 4

Having demonstrated the lack of effect on the skin in its natural state(Example 3), a series of chemical compounds was evaluated foreffectiveness in absorbing the light energy and then transferring thisabsorbed energy, via conduction, into or through the targeted tissue ofthe stratum corneum. Compounds tested included India ink; “SHARPIE”brand indelible black, blue, and red marking pens; methylene blue;fuschian red; epolite #67, an absorbing compound developed for moldinginto polycarbonate lenses for protected laser goggles; tincture ofiodine; iodine-polyvinylpyrrolidone complex (“BETADINE”); copperphthalocyanine; and printers ink.

Using both of the CW laser diodes described in Example 2, positiveablation results were observed on the in vitro samples of heat-separatedstratum corneum prepared according to Example 1 when using all of theseproducts, however some performed better than others. In particular thecopper phthalocyanine (CPC) and the epolite #67 were some of the mosteffective. One probable reason for the superior performance of the CPCis its high boiling point of greater the 500° C. and the fact that itmaintains its solid phase up to this temperature.

Example 5

As copper phthalocyanine has already been approved by the FDA for use inimplantable sutures, and is listed in the Merck index as a rather benignand stabile molecule in regard to human biocompatability, the next steptaken was to combine the topical application of the CPC and the focusedlight source to the skin of healthy human volunteers. A suspension offinely ground CPC in isopropyl alcohol was prepared. The method ofapplication used was to shake the solution and then apply a small dropat the target site. As the alcohol evaporated, a fine and uniformcoating of the solid phase CPC was then left on the surface of the skin.

The apparatus show in FIG. 1 was then applied to the site, wherein theCPC had been topically coated onto the skin, by placing the selectedarea of the individual's skin against a reference plate. The referenceplate consists of a thin glass window roughly 3 cm×3 cm, with a 4 mmhole in the center. The CPC covered area was then positioned such thatit was within the central hole. A confocal video microscope (FIG. 1) wasthen used to bring the surface of the skin into sharp focus. Positioningthe skin to achieve the sharpest focus on the video system alsopositioned it such that the focal point of the laser system wascoincident with the surface of the skin. The operator then activated thepulses of laser light while watching the effects at the target site onthe video monitor. The amount of penetration was estimated visually bythe operator by gauging the amount of defocusing of the laser spot inthe micropore as the depth of the micropore increased, and this can bedynamically corrected by the operator, essentially following the ablatedsurface down into the tissues by moving the position of the camera/lasersource along the “z” axis, into the skin. At the point when the stratumcorneum had been removed down to the epidermis, the appearance of thebase of the hole changed noticeably, becoming much wetter and shinier.Upon seeing this change, the operator deactivated the laser. In manyinstances, depending on the state of hydration of the subject as well asother physiological conditions, a dramatic outflow of interstitial fluidoccurred in response to the barrier function of the stratum corneumbeing removed over this small area. The video system was used to recordthis visual record of the accessibility of interstitial fluid at theporation site.

Example 6

The procedure of Example 5 was followed except that the CPC was appliedto a transparent adhesive tape, which was then caused to adhere to aselected site on the skin of an individual. The results weresubstantially similar to those of Example 5.

Example 7

Histology experiments were performed on cadaver skin according tomethods well known in the art to determine ablation thresholdparameter's for given dye mixtures and collateral damage information.The top surface of the skin sample was treated with a solution of copperphthalocyanine (CPC) in alcohol. After the alcohol evaporated, a topicallayer of solid phase CPC was distributed over the skin surface with amean thickness of 10 to 20 μm. FIG. 8A shows a cross-section of fullthickness skin prior to the laser application, wherein the CPC layer270, stratum corneum 274, and underlying epidermal layers 278 are shown.FIG. 8B shows the sample after a single pulse of 810 nm light wasapplied to an 80 um diameter circle with an energy density of 4000J/cm2, for a pulse period of 20 ms. It is noteworthy that there wasstill a significant amount of CPC present on the surface of the stratumcorneum even in the middle of the ablated crater 282. It should also benoted that laboratory measurements indicate that only about 10% of thelight energy incident on the CPC is actually absorbed, with the other90% being reflected or backscattered. Thus the effective energy fluxbeing delivered to the dye layer which could cause the desired heatingis only about 400 J/cm2. 8C shows the sample after 5 pulses of 810 nmlight were applied, wherein the stratum corneum barrier was removed withno damage to the underlying tissue. These results are a goodrepresentation of the “ideal” optically modulated thermal ablationperformance. FIG. 8D shows the sample after 50 pulses were applied.Damaged tissue 286 was present in the epidermal layers due tocarbonization of non ablated tissue and thermal denaturing of theunderlying tissue. FIGS. 8A-8C show separations between the stratumcorneum and the underlying epidermal layers due to an artifact ofdehydration, freezing, and preparations for imaging.

Example 8

To examine the details of the thermal ablation mechanism, a mathematicalmodel of the skin tissues was constructed upon which various differentembodiments of the thermal ablation method could be tried. This modelcomputes the temperature distribution in a layered semi-infinite mediumwith a specified heat flux input locally on the surface and heat removalfrom the surface some distance away, i.e. convection is applied betweenthe two. The axisymmetric, time-dependent diffusion equation is solvedin cylindrical coordinates using the alternating-direction-implicit(ADI) method. (Note: Constant Temp. B.C. is applied on lower boundary toserve as z->inf; and zero radial heat flux is applied on max radialboundary to serve as r->inf). The layers are parallel to the surface andare defined as: (1) dye; (2) stratum corneum; (3) underlying epidermis;and (4) dermis. The depth into the semi-infinite medium and thermalproperties, density (rho), specific heat (c), and conductivity (k) mustbe specified for each layer.

First, a heat-transfer coefficient, h, on the skin is computed based onthe “steady,” “1-D,” temperature distribution determined by the ambientair temperature, skin surface temperature, and dermis temperature. It isassumed that there is no dye present and provides “h” on the skinsurface. The program then allows one to use this “h” on the dye layersurface or input another desired “h” for the dye surface. Next, the“steady” temperature distribution is computed throughout all layers(including the dye layer) using the specified “h” at the dye surface.This temperature distribution is the initial condition for thetime-dependent heating problem. This constitutes the “m-file” initial.m.The program then solves for the time-dependent temperature distributionby marching in time, computing and displaying the temperature field ateach step.

Each embodiment of the method described herein, for which empirical datahave been collected, has been modeled for at least one set ofoperational parameters, showing how stratum corneum ablation can beachieved in a precise and controllable fashion. The output of thesimulations is presented graphically in two different formats: (1) across-sectional view of the skin showing the different tissue layerswith three isotherms plotted on top of this view which define threecritical temperature thresholds, and (2) two different temperature -vs-time plots, one for the point in the middle of the stratum corneumdirectly beneath the target site, and the second for the point at theboundary of the viable cell layers of the epidermis and the underside ofthe stratum corneum. These plots show how the temperature at each pointvaries with time as the heat pulses are applied as if one could implanta microscopic thermocouple into the tissues. In addition, theapplication of this model allows investigation of the parametric limitswithin which the method can be employed to set the outer limits for twoimportant aspects of the methods performance. First, general cases arepresented cases that define the envelope within which the method can beemployed without causing pain or undesired tissue damage.

For any given heat source, as described in the several differentembodiments of the invention, there is a point at which the effect onthe subject's skin tissues becomes non-optimal in that the subjectperceives a pain sensation, or that the viable cells in the underlyingepidermis and/or dermis sustain temperatures, which if maintained for along enough duration, will render damage to these tissues. Accordingly,a test simulation was run using the optically heated topical copperphthalocyanine (CPC) dye embodiment as a baseline method to establishhow the thermal time constants of the different skin tissue layersessentially define a, window within which the method can be employedwithout pain or damage to adjacent tissue layers.

FIGS. 9 and 10 show schematic cross-sectional views of the skin and thetopical dye layer. In each, figure, three distinct isotherms aredisplayed: (1) 123 C, the point at which vaporization of the water inthe tissue produces an ablation of the tissue; (2) 70 C, the point atwhich viable cells will be damaged if this temperature is maintained forseveral seconds; and (3) 45 C, the average point at which a sensation ofpain will be perceived by the subject. This pain threshold is describedin several basic physiology texts, but experience shows this thresholdto be somewhat subjective. In fact, in repeated tests on the sameindividual, different poration sites within a few millimeters of eachother can show significantly different amounts of sensation, possiblydue to the proximity to a nerve ending in relationship to the porationsite.

The dimensions on the graphs show the different layers of the dye andskin, as measured in m, with flat boundaries defining them. Whereas theactual skin tissues have much more convoluted boundaries, in a meansense for the dimensions involved, the model provides a goodapproximation of the thermal gradients present in the actual tissues.The dimensions used in this, and all subsequent simulations, for thethicknesses of the CPC dye layer and the various skin layers are asfollows: dye, 10 m; stratum corneum, 30 m; underlying epidermis, 70 m;and dermis, 100 m.

Additional conditions imposed on the model for this particularsimulation are shown in the following tables:

TABLE 1 Initial Conditions for Finite Difference Thermal Model AmbientAir Temperature Ta = 20 C. Skin Surface Temperature Ts = 30 C. DermisTemperature Td = 37 C. Dye Vaporization Temperature Tvap = 550 C. S.C.Vaporization Temperature Tc1 = 123 C. Tissue Damage Temperature Tc2 = 70C. “Pain” Temperature Tc3 = 45 C. Radius of Irradiated Area R_(hot) = 30m Energy Density Applied FLUX = 400 Joules/cm²

TABLE 2 Parameter Dye S.C. Epidermis Dermis Thermal 0.00046 .001230.00421 0.00421 Conductivity Density 0.67 1.28 1.09 1.09 Specific Heat0.8 1.88 3.35 3.35

When these simulations are run, the following conservative assumptionsare imposed:

1. While some portion of the stratum corneum may be shown as having atemperature already exceeded the ablation threshold for thermalvaporization of the water content, this event is not modeled, and thesubsequent loss of heat energy in the tissues due to this vaporizationis not factored into the simulation. This will cause a slight elevationin the temperatures shown in the underlying tissues from that point onin the simulation run.

2. Similarly, when some portion of the copper phthalocyanine (CPC) dyelayer is shown to have reached its vaporization point of 550° C., thisevent is not modeled, but the temperature is merely hard-limited to thislevel. This will also cause a slight elevation of the subsequenttemperatures in the underlying layers as the simulation progresses.

Even with these simplifications used in the model, the correlationbetween the predicted performance and the empirically observedperformance based on both clinical studies and histological studies ondonor tissue samples is remarkable. The key data to note in FIGS. 9 and10 are the length of time which the heat pulse is applied, and thelocation of the three different threshold temperatures displayed by theisotherms.

In FIG. 9, with a pulse length of 21 milliseconds, the 70° C. isothermjust crosses the boundary separating the stratum corneum and the viablecell layers in the epidermis. In in vitro studies on donor skin samplesunder these conditions, fifty pulses of thermal energy delivered 50milliseconds apart cause detectable damage to this top layer of livingcells (see FIG. 8D). However, it was also shown in the in vitro studiesthat five pulses of heat energy at these same operating parameters, didnot produce any significant damage to these tissues. It seems reasonablethat even though the nominal damage threshold may have been exceeded, atleast in a transient sense, this temperature must be maintained for somecumulative period of time to actually cause any damage to the cells.Nevertheless, the basic information presented by the simulation is thatif one keeps the “on-time” of the heat pulse to less than 20milliseconds with the flux density of 400 Joules/cm², then no damage tothe living cells in the underlying epidermis will be sustained, eventhough the ablation threshold isotherm has been moved well into orthrough the stratum corneum. In other words, by using a low flux densitythermal energy source, modulated such that the “on time” is suitablyshort, ablation of the stratum corneum can be achieved without anydamage to the adjacent cells in the underlying epidermis (see FIG. 8C).This is possible in large part due to the significantly differentthermal diffusivities of these two tissues layers. That is, the stratumcorneum, containing only about 10% to 20% water content, has a muchlower thermal conductivity constant, 0.00123 J/(S m*K), than the 0.00421J/(S*cm*K) of the epidermis. This allows the temperature to build up inthe stratum corneum, while maintaining a tight spatial definition, tothe point at which ablation will occur.

In FIG. 10, the same simulation scenario started in the damage thresholdcritical point run illustrated in FIG. 9 is carried out farther in time.By leaving the heat pulse on for 58 milliseconds at the same fluxdensity of 400 Joules/cm² within the 60 μm diameter circle of dye beingheated, the pain sensory isotherm at 45° C. just enters the innervatedlayer of skin comprised by the dermis. In addition, the damage thresholdisotherm moves significantly farther into the epidermal layer than whereit was shown to be in FIG. 9. Relating this simulation to the numerousclinical studies conducted with this method, an excellent verificationof the model's accuracy is obtained in that the model shows almostexactly the duration of ‘on-time’ that the heat probe can be applied tothe skin before the individual feels it. In clinical tests, acontrollable pulse generator was used to set the “on-time” and“off-time” of a series of light pulses applied to the topical layer ofcopper phthalocyanine (CPC) dye on the skin. While maintaining aconstant “off-time” of 80 milliseconds, the “on-time” was graduallyincreased until the subject reported a mild “pain” sensation. Withoutexception, all of the subjects involved in these studies, reported thefirst “pain” at an “on-time” of between 45 and 60 milliseconds, veryclose to that predicted by the model. In addition, the site-to-sitevariability mentioned previously as regards the sensation of “pain” wasnoted in these clinical studies. Accordingly, what is reported as “pain”is the point at which the first unambiguous sensation is noticeable. Atone site this may be reported as pain, whereas at an adjacent site thesame subject may report this as merely “noticeable.”

One element of this clinical research is the realization that even atthe same site, a non-uniform pulse-train of heat pulses may work withthe subject's psycho-physiological neuro-perception to cause a genuinereduction in perceived sensation. For example, a series of shorterlength heat pulses can be used to saturate the neurons in the area,momentarily depleting the neuro-transmitters available at this synapticjunction and therefore limiting the ability to send a “pain” message.This then allows a longer pulse following these short pulses to be lessnoticeable than if it were applied at the beginning of the sequence.Accordingly, a series of experiments was conducted with some arbitrarilycreated pulse trains, and the results were consistent with thishypothesis. An analogy for this situation might be found in theperception when one first steps into a very hot bath that is painful atfirst, but quickly becomes tolerable as one acclimates to the heatsensation.

Example 9

An object of this invention is to achieve a painless, micro-poration ofthe stratum corneum without causing any significant damage to theadjacent viable tissues. As described in the simulation illustrated inExample 8 and FIGS. 9-10, a boundary appears to exist for any given fluxdensity of thermal energy within the ablation target spot within whichthe micro-poration can be achieved in just such a painless andnon-traumatic manner. Both the in vivo and in vitro studies have shownthat this is the case, and this has permitted development throughempirical methods of some operational parameters that appear to workvery well. The following set of simulations shows how the method workswhen these specific parameters are used.

In the first case, a pulse train of ten pulses, 10 milliseconds“on-time” separated by 10 milliseconds “off-time” is applied to theCPC-covered skin. FIG. 11 shows the final temperature distribution inthe skin tissues immediately after this pulse train has ended. As can beseen, the isotherms representing the three critical temperaturethresholds show that stratum corneum ablation has been achieved, with nosensation present in the dermal layer nerves and very little cross-overof the damage threshold into or through the viable cells of theunderlying epidermis. As mentioned previously, it appears that toactually do permanent cell damage, the epidermal cells must not only beheated up to a certain point, but they also must be held at thistemperature for some period of time, generally thought to be about fiveseconds. FIGS. 12 and 13 show the temperature of the stratum corneum andthe viable epidermis, respectively, as a function of time, showingheating during the “on-time” and cooling during the “off-time” for theentire ten cycles. Relating this simulation to the in vivo studiesconducted, note that in better than 90% of the poration attempts withthe system parameters set to match the simulation, effective poration ofthe stratum corneum was achieved without pain to the subject, and insubsequent microscopic examination of the poration site several dayslater, no noticeable damage to the tissues was apparent. The in vitrostudies conducted on whole thickness donor skin samples were alsoconsistent with the model's prediction of behavior.

Example 10

In conducting both the empirical in vivo studies, and these simulations,it appears that prechilling of the skin aids in optimizing themicro-poration process for reducing the probability of pain or damage toadjacent tissues. In practice, this can easily be achieved using asimple cold-plate placed against the skin prior to the poration process.For example, applying a Peltier cooled plate to the 1 cm diameter circlesurrounding the poration target site, with the plate held at roughly 5°C. for a few seconds, significantly reduces the temperature of thetissues. A schematic illustration of an experimental device used forthis purpose in the laboratory is shown in FIGS. 3A-B. By applyingexactly the same ten-cycle pulse train as used in the run illustrated inExample 9, one can see, by comparing FIG. 11 to FIG. 14, FIG. 12 to FIG.15, and FIG. 13 to FIG. 16, how much improvement can be made in thecontrol of the temperature penetration into or through the skin tissues.Once again, the relatively low thermal diffusivity and specific heat ofthe stratum corneum as compared to the epidermis and dermis isadvantageous. Once cooled, the highly hydrated tissues of the epidermisand dermis require a much larger thermal energy input to elevate theirtemperatures, whereas the stratum corneum, with its relatively drymakeup, can quickly be heated up to the ablation threshold.

Example 11

Once the basic thermal conduction mechanism of delivering the energyinto or through the skin tissues underlying the effective painlessablation and micro-poration of the stratum corneum is understood,several different specific methods to achieve the required rapidtemperature modulations of the contact point can be conceived, such asthe hot wire embodiments illustrated in FIGS. 4-7.

A basic embodiment, as described herein, uses an Ohmic heating element(FIG. 4), such as the tip of a small cordless soldering iron, with asuitably sized, relatively non-reactive, wire wrapped around it with ashort amount of the wire left to protrude away from the body of theheater. When electricity is applied with a constant current source, theheater will come up to some temperature and within a few seconds,achieve a steady state with the convection losses to the surroundingair. Similarly, the wire, which is a part of this thermal system, willreach a steady state such that the very tip of the wire can be raised toalmost any arbitrary temperature, up to roughly 1000° C. with thesetypes of components. The tip can be sized to give exactly the dimensionmicropore desired.

In the laboratory, tungsten wires with a diameter of 80 μm attached tothe replaceable tip of a “WAHL” cordless soldering iron withapproximately 2 mm of wire protruding from the tip have been utilized.With a thermocouple, the temperature of the tip has been measured at itssteady state, and it has been noted that by varying the constant currentsettings, steady state temperatures of greater than 700° C. can easilybe reached. To achieve the desired modulation, a low mass, fast responseelectromechanical actuator was coupled to the tip such that the positionof the wire could be translated linearly more than 2 mm at up to a 200Hz rate. Then, by mounting the entire apparatus on a precision stage,this vibrating tip could very controllably be brought into contact withthe skin surface in a manner where it was only in contact for less than10 milliseconds at a time, the “on-time,” while an “off-time” ofarbitrarily long periods could be achieved by setting the pulsegenerator accordingly. These in vivo studies showed that the porationcould actually be achieved before the subject being porated even knewthat the tip of the wire was being brought into contact with the skin.

To compare the performance of this embodiment to the optically heatedtopical CPC dye embodiment, the following simulations were run accordingto the procedure of Example 8. Essentially, by only varying the initialconditions, the hot wire embodiment can be run with the identicalsimulation code. Because the contact with the wire occurs essentiallyinstantly, there is no time dependent build-up of heat in the CPC dyelayer and when the wire is physically removed from contact with theskin, there is a no residual heat still left on the surface as there iswith the heated CPC dye layer. Also, as the wire itself defines the areatargeted for ablation/micro-poration, there should be no lateraldiffusion of thermal energy prior to its application to the stratumcorneum. The comparative performances of the “hot-wire” embodiment areshown in FIGS. 17-19.

Example 12

In this example, the procedure of Example 11 was followed except thatthe skin was pre-cooled according to the procedure of Example 10.Similarly, pre-cooling the target site yields similarly positive resultswith the “hot-wire” embodiment. The results of the pre-cooled simulationof the “hot-wire” approach are shown in FIGS. 20-22.

Example 13

As discussed in the background introduction of this disclosure, theTankovich '803 patent appears at first glance to be similar to thepresently claimed invention. In this example, the simulation model wasset up with the operating parameters specified in Tankovich '803, i.e. apulse width of 1 s and a power level of 40,000,000 W/cm². FIGS. 23 and24 show that under these conditions no portion of the stratum corneumreaches the threshold for flash vaporization of water, 123 C, and thusno ablation/microporation of the stratum corneum occurs. In practice,applying this type of high peak power, short duration pulse to thetopical dye layer merely vaporizes the dye off of the surface of theskin with no effect on the skin. This example, thus, demonstrates thatthe conditions specified by Tankovich '803 are inoperative in thepresently claimed invention.

Example 14

In this example, interstitial fluid obtained after porating the skinaccording to the procedure of Example 6 was collected and analyzed todetermine the glucose concentration thereof. Data were obtained on fournon-diabetic subjects and six type I diabetic subjects undergoing aglucose load test. Subject's ages ranged from 27 to 43. The goal of thestudy was to examine the utility of the method for painlessly harvestingenough interstitial fluid (ISF) from the subjects to allow the ISFsamples to be assayed for glucose content, and then compare theseconcentrations to the glucose level presenting in the subject's wholeblood.

All subjects had both the blood and ISF glucose assays performed withthe “ELITE” system from Miles-Bayer. All ten subjects underwentidentical measurement protocols, with adjustments being made regardingthe glucose load and insulin shot for those subjects with insulindependent diabetes.

The basic design of the study was to recruit a modest number ofvolunteers, some with diabetes and some without diabetes, from which aseries of sample pairs of ISF and whole blood were drawn every 3 to 5minutes throughout the 3 to 4 hour duration of the study period. Boththe blood and the ISF samples were assayed for glucose and thestatistical relationship between the blood glucose levels and theinterstitial fluid determined. To examine the hypothesized temporal lagof the ISF glucose levels as compared to the whole blood glucose levels,the study subjects were induced to exhibit a significant and dynamicchange in their glucose levels. This was accomplished by having eachsubject fast for 12 hours prior to beginning the test and then givingthe subject a glucose load after his or her baseline glucose levels havebeen established via a set of three fasting blood and ISF glucoselevels. After the baseline levels had been established, the subjectswere given a glucose load in the form of sweet juice based on thefollowing guidelines:

-   -   i. For the control subjects, the glucose load was calculated        based on a 0.75 gram glucose per pound of body weight.    -   ii. For the subjects with insulin dependent diabetes the glucose        load was 50 grams of glucose. In addition, immediately after        taking the glucose load the diabetic subjects will self inject        their normal morning dose of fast acting insulin. In the case        where the diabetic subject presents with fasting glucose levels        above 300 mg/dL, they were asked to give themselves their        insulin injection first, and the glucose load was provided after        their blood glucose levels have dropped to below 120 mg/dL.

Each subject recruited was first given a complete description of thestudy in the “Informed Consent” document which they were required tounderstand and sign before they were officially enrolled into theprogram. Upon acceptance, they completed a medical historyquestionnaire. The detailed clinical procedure implemented was:

(a) Subject fasted from 9:00 p.m. the night before the study visit,consuming only water. No caffeine, cigarettes, fruit juice were allowedduring this period.

(b) Subject arrived at the testing facility by 9:00 a.m. the next day.

(c) Subject was seated in a reclining chair provided for the subject torelax in throughout the study procedure.

(d) Both whole blood and ISF samples were taken at three to five minuteintervals beginning upon the subject's arrival and continuing for thenext three to four hours. The duration over which the data werecollected was based on when the subject's blood glucose levels hadreturned to the normal range and stabilized after the glucose load. TheISF samples were harvested using the optical poration, ISF pumpingmethod, described in more detail below. Each ISF sample was roughly 5 μLby volume to ensure a good fill of the ELITE test strip. The bloodsamples were obtained via a conventional finger prick lancet. Both theISF and the blood samples were immediately assayed for glucose with theELITE home glucometer system from Miles-Bayer. To improve the estimateof the ‘true’ blood glucose levels, two separate ELITE assays were bedone on each finger stick sample.

(e) To facilitate the continued collection of the ISF from the same sitethrough-out the entire data collection phase for a given individual, a 5by 5 matrix of twenty five micropores was created on the subject's upperforearm, each micropore being between 50 and 80 μm across and spaced 300μm apart. A 30 μm diameter teflon disk with a 6 mm hole in the centerwas attached to the subject's forearm with a pressure sensitive adhesiveand positioned such that the 6 mm center hole was located over the 5 by5 matrix of micropores. This attachment allowed a convenient method bywhich a small suction hose could be connected, applying a mild vacuum(10 to 12 inches of Hg) to the porated area to induce the ISF to flowout of the body through the micropores. The top of the teflon disk wasfitted with a clear glass window allowing the operator to directly viewthe micro-porated skin beneath it. When a 5 μL bead of ISF was formed onthe surface of the skin, it could easily be ascertained by visuallymonitoring the site through this window. This level of vacuum created anominal pressure gradient of around 5 pounds/square inch (PSI). Withoutthe micropores, no ISF whatsoever could be drawn from the subject's bodyusing only the mild vacuum.

(f) After the first three sample pairs have been drawn, the subject wasgiven a glucose load in the form of highly sweetened orange juice. Theamount of glucose given was 0.75 grams per pound of body weight for thenondiabetic subjects and 50 grams for the diabetic subjects. Thediabetic subjects also self administered a shot of fast acting insulin,(regular) with the dosage appropriately calculated, based on this 50gram level of glucose concurrent with the ingestion of the glucose load.With the normal 1.5 to 2.5 hour lag between receiving an insulin shotand the maximum effect of the shot, the diabetic subjects were expectedto exhibit an upwards excursion of their blood glucose levels ranging upto 300 mg/dL and then dropping rapidly back into the normal range as theinsulin takes effect. The nondiabetic subjects were expected to exhibitthe standard glucose tolerance test profiles, typically showing a peakin blood glucose levels between 150 mg/dL and 220 mg/dL from 45 minutesto 90 minutes after administering the glucose load, and then a rapiddrop back to their normal baseline levels over the next hour or so.

(g) Following the administration of the glucose load or glucose load andinsulin shot, the subjects had samples drawn, simultaneously, of ISF andfinger prick whole blood at five minute intervals for the next three tofour hours. The sampling was terminated when the blood glucose levels inthree successive samples indicate that the subject's glucose hadstabilized.

Upon examination of the data, several features were apparent. Inparticular, for any specific batch of ELITE test strips, there exist adistinct shift in the output shown on the glucometer in mg/dL glucose ascompared to the level indicated on the blood. An elevated reading wouldbe expected due to the lack of hematocrit in the ISF and to the normaldifferences in the electrolyte concentrations between the ISF and wholeblood. Regardless of the underlying reasons for this shift in output, itwas determined via comparison to a reference assay that the true ISFglucose levels are linearly related to the values produced by the ELITEsystem, with the scaling coefficients constant for any specific batch ofELITE strips. Consequently, for the comparison of the ISF glucose levelsversus the whole blood measurements, first order linear correction wasapplied to the ISF data as follows:

ISF_(glucose)=0.606ISF_(ELITE)+19.5

This scaling of the output of the ELITE glucometer when used to measureISF glucose levels, allows one to examine, over the entire data set, theerror terms associated with using ISF to estimate blood glucose levels.Of course, even with no linear scaling whatsoever, the correlationsbetween the ISF glucose values and the blood glucose levels are the sameas the scaled version.

Based on the majority of the published body of literature on the subjectof ISF glucose as well as preliminary data, it was originally expectedthat a 15 to 20 minute lag between the ISF glucose levels and the thosepresented in the whole blood from a finger stick would be observed. Thisis not what the data showed when analyzed. Specifically, when eachindividual's data set is analyzed to determine the time shift requiredto achieve the maximum correlation between the ISF glucose levels andthe blood glucose levels it was discovered that the worst case time lagfor this set of subjects was only 13 minutes and the average time lagwas only 6.2 minutes, with several subjects showing a temporal trackingthat was almost instantaneous (about 1 minute).

Based on the minimal amount of lag observed in this data set, the graphshown in FIG. 25 presents all ten of the glucose load tests,concatenated one after another on an extended time scale. The data arepresented with no time shifting whatsoever, showing the high level oftracking between the ISF and blood glucose levels the entire clinicaldata set being dealt with in exactly the same manner. If the entire dataset is shifted as a whole to find the best temporal tracking estimate,the correlation between the ISF and blood glucose levels peaks with adelay of two (2) minutes at an r value of r=0.97. This is only a trivialimprovement from the unshifted correlation of r=0.964. Therefore, forthe remainder of the analysis the ISF values are treated with no timeshift imposed on them. That is, each set of blood and ISF glucose levelsis dealt with as simultaneously collected data pairs.

After the unshifted Elite ISF readings had been scaled to reflect theproportional glucose present in the ISF, it was possible to examine theerror associated with these data. The simplest method for this is toassume that the average of the two ELITE finger-stick blood glucosereadings is in fact the absolutely correct value, and then to merelycompare the scaled ISF values to these mean blood glucose values. Thesedata are as follows: Standard Deviation Blood-ISF, 13.4 mg/dL;Coefficient of Variance of ISF, 9.7%; Standard Deviation of the TwoElites, 8.3 mg/dL; and Coefficient of Variance of Blood (Miles), 6%.

As these data show, the blood based measurement already contains anerror term. Indeed, the manufacturer's published performance dataindicates that the ELITE system has a nominal Coefficient of Variance(CV) of between 5% and 7%, depending on the glucose levels and theamount of hematocrit in the blood.

An additional look at the difference term between the ISF glucose andthe blood glucose is shown in the form of a scatter plot in FIG. 26. Inthis figure, the upper and lower bounds of the 90% confidence intervalare also displayed for reference. It is interesting to note that withonly two exceptions, all of the data in the range of blood glucoselevels below 100 mg/dL fall within these 90% confidence interval errorbars. This is important as the consequences of missing a trend towardshypoglycemia would be very significant to the diabetic user. That is, itwould be much better to under-predict glucose levels in the 40 to 120mg/dL than to over predict them.

Essentially, if one assumes that the basic assay error when the ELITEsystem is used on ISF is comparable to the assay error associated withthe ELITE's use on whole blood, then the Deviation of the ISF glucosefrom the blood glucose can be described as:

ISF_(deviation)=[(ISF_(actual))²+(ISF_(actual))²]^(1/2).

Applying this equation to the values shown above, one can solve for theestimated ‘true’ value of the ISF error term:

ISF_(actual)=[(ISF_(deviation))²−(Blood_(actual))²]^(1/2).

Or, solving the equation,

ISF_(actual)=[(13.4)²−(8.3)²]^(1/2)=10.5 mg/dl.

A histogram of the relative deviation of the ISF to the blood glucoselevels is shown in FIG. 27.

Drug Delivery Through Pores in the Biological Membrane

The present invention also includes a method for the delivery of drugs,including drugs currently delivered transmembrane, through micropores inthe stratum corneum or other biological membrane. In one illustrativeembodiment, the delivery is achieved by placing the solution in areservoir over the poration site. In another illustrative embodiment, apressure gradient is used to further enhance the delivery. In stillanother illustrative embodiment, sonic energy is used with or without apressure gradient to further enhance the delivery. The sonic energy canbe operated according to traditional transdermal parameters or byutilizing acoustic streaming effects, which will be describedmomentarily, to push the delivery solution through the poratedbiological membrane.

Example 15

This example shows the use of stratum corneum poration for the deliveryof lidocaine, a topical analgesic. The lidocaine solution also containeda chemical permeation enhancer formulation designed to enhance itspassive diffusion across the stratum corneum. A drawing of anillustrative delivery apparatus 300 is shown in FIG. 28, wherein theapparatus comprises a housing 304 enclosing a reservoir 308 for holdinga drug-containing solution 312. The top portion of the housing comprisesan ultrasonic transducer 316 for providing sonic energy to aid intransporting the drug-containing solution through micropores 320 in thestratum corneum 324. A port 328 in the ultrasonic transducer permitsapplication of pressure thereto for further aiding in transporting thedrug-containing solution through the micropores in the stratum corneum.The delivery apparatus is applied to a selected area of an individual'sskin such that it is positioned over at least one, and preferably aplurality, of micropores. An adhesive layer 332 attached to a lowerportion of the housing permits the apparatus to adhere to the skin suchthat the drug-containing solution in the reservoir is in liquidcommunication with the micropores. Delivery of the drug through themicropores results in transport into the underlying epidermis 336 anddermis 340.

Five subjects were tested for the effectiveness of drug delivery usingporation together with ultrasound. The experiment used two sites on thesubjects left forearm about three inches apart, equally spaced betweenthe thumb and upper arm. The site near the thumb will be referred to assite 1 the site furthest from the thumb will be referred to as site 2.Site 1 was used as a control where the lidocaine and enhancer solutionwas applied using an identical delivery apparatus 300, but without anymicro-poration of the stratum corneum or sonic energy. Site 2 wasporated with 24 holes spaced 0.8 millimeters apart in a grid containedwithin a 1 cm diameter circle. The micropores in Site 2 were generatedaccording to the procedure of Example 6. Lidocaine and low levelultrasound were applied. Ultrasound applications were made with a custommanufactured Zevex ultrasonic transducer assembly set in burst mode with0.4 Volts peak to peak input with 1000 count bursts occurring at 10 Hzwith a 65.4 kHz fundamental frequency, i.e., a pulse modulated signalwith the transducer energized for 15 millisecond bursts, and then turnedoff for the next 85 milliseconds. The measured output of the amplifierto the transducer was 0.090 watts RMS.

After application of the lidocaine, sensation measurements were made byrubbing a 30 gauge wire across the test site. Experiments were executedon both sites, Site 1 for 10 to 12 minute duration and Site 2 for two 5minute duration intervals applied serially to the same site. Both siteswere assessed for numbness using a scale of 10 to 0, where 10 indicatedno numbness and 0 indicated complete numbness as reported by the testsubjects. The following summary of results is for all 5 subjects.

The control site, site 1, presented little to no numbness (scale 7 to10) at 10 to 12 minutes. At approximately 20 minutes some numbness(scale 3) was observed at site 1 as the solution completely permeatedthe stratum corneum. Site 1 was cleaned at the completion of thelidocaine application. Site 2 presented nearly complete numbness (scale0 to 1) in the 1 cm circle containing the porations. Outside the 1 cmdiameter circle the numbness fell off almost linearly to 1 at a 2.5 cmdiameter circle with no numbness outside the 2.5 cm diameter circle.Assessment of site 2 after the second application resulted in a slightlylarger totally numb circle of about 1.2 cm diameter with numbnessfalling off linearly to 1 in an irregular oval pattern with a diameterof 2 to 2.5 cm perpendicular to the forearm and a diameter of 2 to 6 cmparallel to the forearm. Outside the area no numbness was noted. Agraphic representation of illustrative results obtained on a typicalsubject is shown in FIGS. 29A-C. FIGS. 29A and 29B show the resultsobtained at Site 2 (porated) after 5 and 10 minutes, respectively. FIG.29C shows the results obtained at Site 1 (control with no poration).

Sonic Energy and Enhancers for Enhancing Transdermal Flux

The physics of sonic energy fields created by sonic transducers can beutilized in a method by which sonic frequency can be modulated toimprove on flux rates achieved by other methods. As shown in FIG. 1 ofU.S. Pat. No. 5,445,611, hereby incorporated herein by reference, theenergy distribution of an sonic transducer can be divided into near andfar fields. The near field, characterized by length N, is the zone fromthe first energy minimum to the last energy maximum. The zone distal tothe last maximum is the far field. The near (N) field pattern isdominated by a large number of closely spaced local pressure peaks andnulls. The length of the near field zone, N, is a function of thefrequency, size, and shape of the transducer face, and the speed ofsound in the medium through which the ultrasound travels. For a singletransducer, intensity variations within its normal operating range donot affect the nature of the sonic energy distribution other than in alinear fashion. However, for a system with multiple transducers, allbeing modulated in both frequency and amplitude, the relativeintensities of separate transducers do affect the energy distribution inthe sonic medium, regardless of whether it is skin or another medium.

By changing the frequency of the sonic energy by a modest amount, forexample in the range of about 1 to 20%, the pattern of peaks and nullsremains relatively constant, but the length N of the near field zonechanges in direct proportion to the frequency. Major changes thefrequency, say a factor of 2 or more, will most likely produce adifferent set of resonances or vibrational modes in the transducer,causing a significantly and unpredictably different near field energypattern. Thus, with a modest change in the sonic frequency, the complexpattern of peaks and nulls is compressed or expanded in anaccordion-like manner. By selecting the direction of frequencymodulation, the direction of shift of these local pressure peaks can becontrolled. By applying sonic energy at the surface of the skin,selective modulation of the sonic frequency controls movement of theselocal pressure peaks through the skin either toward the interior of thebody or toward the surface of the body. A frequency modulation from highto low drives the pressure peaks into the body, whereas a frequencymodulation from low to high pulls the pressure peaks from within thebody toward the surface and through the skin to the outside of the body.

Assuming typical parameters for this application of, for example, a 1.27cm diameter sonic transducer and a nominal operating frequency of 10 MHzand an acoustic impedance similar to that of water, a frequencymodulation of 1 MHz produces a movement of about 2.5 mm of the peaks andnulls of the near field energy pattern in the vicinity of the stratumcorneum. From the perspective of transdermal and/or transmucosalwithdrawal of analytes, this degree of action provides access to thearea well below the stratum corneum and even the epidermis, dermis, andother tissues beneath it. For any given transducer, there may be anoptimal range of frequencies within which this frequency modulation ismost effective.

The flux of a drug or analyte across the skin can also be increased bychanging either the resistance (the diffusion coefficient) or thedriving force (the gradient for diffusion). Flux can be enhanced by theuse of so-called penetration or chemical enhancers.

Chemical enhancers are comprised of two primary categories ofcomponents, i.e., cell-envelope disordering compounds and solvents orbinary systems containing both cell-envelope disordering compounds andsolvents.

Cell envelope disordering compounds are known in the art as being usefulin topical pharmaceutical preparations and function also in analytewithdrawal through the skin. These compounds are thought to assist inskin penetration by disordering the lipid structure of the stratumcorneum cell-envelopes. A comprehensive list of these compounds isdescribed in European Patent Application 43,738, published Jun. 13,1982, which is incorporated herein by reference. It is believed that anycell envelope disordering compound is useful for purposes of thisinvention.

Suitable solvents include water; diols, such as propylene glycol andglycerol; mono-alcohols, such as ethanol, propanol, and higher alcohols;DMSO; dimethylformamide; N,N-dimethylacetamide; 2-pyrrolidone;N-(2-hydroxyethyl)pyrrolidone, N-methylpyrrolidone,1-dodecylazacycloheptan-2-one and othern-substituted-alkyl-azacycloalkyl-2-ones (azones) and the like.

U.S. Pat. No. 4,537,776, Cooper, issued Aug. 27, 1985, contains anexcellent summary of prior art and background information detailing theuse of certain binary systems for permeant enhancement. Because of thecompleteness of that disclosure, the information and terminologyutilized therein are incorporated herein by reference.

Similarly, European Patent Application 43,738, referred to above,teaches using selected diols as solvents along with a broad category ofcell-envelope disordering compounds for delivery of lipophilicpharmacologically-active compounds. Because of the detail in disclosingthe cell-envelope disordering compounds and the diols, this disclosureof European Patent Application 43,738 is also incorporated herein byreference.

A binary system for enhancing metoclopramide penetration is disclosed inUK Patent Application GB 2,153,223 A, published Aug. 21, 1985, andconsists of a monovalent alcohol ester of a C8-32 aliphaticmonocarboxylic acid (unsaturated and/or branched if C18-32) or a C6-24aliphatic monoalcohol (unsaturated and/or branched if C14-24) and anN-cyclic compound such as 2-pyrrolidone, N-methylpyrrolidone and thelike.

Combinations of enhancers consisting of diethylene glycol monoethyl ormonomethyl ether with propylene glycol monolaurate and methyl laurateare disclosed in U.S. Pat. No. 4,973,468 as enhancing the transdermaldelivery of steroids such as progesterons and estrogens. A dual enhancerconsisting of glycerol monolaurate and ethanol for the transdermaldelivery of drugs is shown in U.S. Pat. No. 4,820,720. U.S. Pat. No.5,006,342 lists numerous enhancers for transdermal drug administrationconsisting of fatty acid esters or fatty alcohol ethers of C₂ to C₄alkanediols, where each fatty acid/alcohol portion of the ester/ether isof about 8 to 22 carbon atoms. U.S. Pat. No. 4,863,970 showspenetration-enhancing compositions for topical application comprising anactive permeant contained in a penetration-enhancing vehicle containingspecified amounts of one or more cell-envelope disordering compoundssuch as oleic acid, oleyl alcohol, and glycerol esters of oleic acid; aC₂ or C₃ alkanol and an inert diluent such as water.

Other chemical enhancers, not necessarily associated with binary systemsinclude DMSO or aqueous solutions of DMSO such as taught in Herschler,U.S. Pat. No. 3,551,554; Herschler, U.S. Pat. No. 3,711,602; andHerschler, U.S. Pat. No. 3,711,606, and the azones(n-substituted-alkyl-azacycloalkyl-2-ones) such as noted in Cooper, U.S.Pat. No. 4,557,943.

Some chemical enhancer systems may possess negative side effects such astoxicity and skin irritation. U.S. Pat. No. 4,855,298 disclosescompositions for reducing skin irritation caused by chemical enhancercontaining compositions having skin irritation properties with an amountof glycerin sufficient to provide an anti-irritating effect.

Because the combination of microporation of the stratum corneum and theapplication of sonic energy accompanied by the use of chemical enhancerscan result in an improved rate of analyte withdrawal or permeantdelivery through the stratum corneum, the specific carrier vehicle andparticularly the chemical enhancer utilized can be selected from a longlist of prior art vehicles some of which are mentioned above andincorporated herein by reference. To specifically detail or enumeratethat which is readily available in the art is not thought necessary. Theinvention is not drawn to the use of chemical enhancers per se and it isbelieved that all chemical enhancers, useful in the delivery of drugsthrough the skin, will function with dyes in optical microporation andalso with sonic energy in effecting measurable withdrawal of analytesfrom beneath and through the skin surface or the delivery of permeantsor drugs through the skin surface.

Example 16

Modulated sonic energy and chemical enhancers were tested for theirability to control transdermal flux on human cadaver skin samples. Inthese tests, the epidermal membrane had been separated from the humancadaver whole skin by the heat-separation method of Example 1. Theepidermal membrane was cut and placed between two halves of thepermeation cell with the stratum corneum facing either the upper (donor)compartment or lower (receiver) compartment. Modified Franz cells wereused to hold the epidermis, as shown in FIG. 2 of U.S. Pat. No.5,445,611. Each Franz cell consists of an upper chamber and a lowerchamber held together with one or more clamps. The lower chamber has asampling port through-which materials can be added or removed. A sampleof stratum corneum is held between the upper and lower chambers whenthey are clamped together. The upper chamber of each Franz cell ismodified to allow an ultrasound transducer to be positioned within 1 cmof the stratum corneum membrane. Methylene blue solution was used as anindicator molecule to assess the permeation of the stratum corneum. Avisual record of the process and results of each experiment was obtainedin a time stamped magnetic tape format with a video camera and videocassette recorder (not shown). Additionally, samples were withdrawn formeasurement with an absorption spectrometer to quantitate the amount ofdye which had traversed the stratum corneum membrane during anexperiment. Chemical enhancers suitable for use could vary over a widerange of solvents and/or cell envelope disordering compounds as notedabove. The specific enhancer utilized was:ethanol/glycerol/water/glycerol monooleate/methyl laurate in50/30/15/2.5/2.5 volume ratios. The system for producing and controllingthe sonic energy included a programmable 0-30 MHz arbitrary waveformgenerator (Stanford Research Systems Model DS345), a 20 watt 0-30 MHzamplifier, and two unfocused ultrasound immersion transducers havingpeak resonances at 15 and 25 MHz, respectively. Six cells were preparedsimultaneously for testing of stratum corneum samples from the samedonor. Once the stratum corneum samples were installed, they wereallowed to hydrate with distilled water for at least 6 hours before anytests were done.

Example 17

Effects of Sonic Energy without Chemical Enhancers

As stated above in Example 16, the heat-separated epidermis was placedin the Franz cells with the epidermal side facing up, and the stratumcorneum side facing down, unless noted otherwise. The lower chamberswere filled with distilled water, whereas the upper chambers were filledwith concentrated methylene blue solution in distilled water.

Heat Separated Epidermis: Immediately after filling the upper chamberswith methylene blue solution, sonic energy was applied to one of thecells with the transducer fully immersed. This orientation wouldcorrespond, for example, to having the transducer on the opposite sideof a fold of skin, or causing the sonic energy to be reflected off areflector plate similarly positioned and being used to “push” analyteout of the other side of the fold into a collection device. The sonicenergy setting was initially set at the nominal operating frequency of25 MHz with an intensity equivalent to a 20 volt peak-to-peak (P-P)input wave form. This corresponds to roughly a 1 watt of average inputpower to the transducer and similarly, assuming the manufacturer'snominal value for conversion. efficiency of 1% for this particulartransducer, a sonic output power of around 0.01 watts over the 0.78 cm²surface of the active area or a sonic intensity of 0.13 watts/cm². Threeother control cells had no sonic energy applied to them. After 5 minutesthe sonic energy was turned off. No visual indication of dye flux acrossthe stratum corneum was observed during this interval in any of thecells, indicating levels less than approximately 0.0015% (v/v) of dyesolution in 2 ml of receiver medium.

Testing of these same 3 control cells and 1 experimental cell wascontinued as follows. The intensity of sonic energy was increased to themaximum possible output available from the driving equipment of a 70volt peak-to-peak input 12 watts average power input or (0.13 watts/cm²)of sonic output intensity. Also, the frequency was set to modulate orsweep from 30 MHz to 10 MHz. This 20 MHz sweep was performed ten timesper second, i.e., a sweep rate of 10 Hz. At these input power levels, itwas necessary to monitor the sonic energy transducer to avoidoverheating. A contact thermocouple was applied to the body of thetransducer and power was cycled on and off to maintain maximumtemperature of the transducer under 42 C. After about 30 minutes ofcycling maximum power at about a 50% duty cycle of 1 minute on and 1minute off, there was still no visually detectable permeation of thestratum corneum by the methylene blue dye.

A cooling water jacket was then attached to the sonic energy transducerto permit extended excitation at the maximum energy level. Using thesame 3 controls and 1 experimental cell, sonic energy was applied atmaximum power for 12 hours to the experimental cell. During this timethe temperature of the fluid in the upper chamber rose to only 35 C,only slightly above the approximately 31° C. normal temperature of thestratum corneum in vivo. No visual evidence of dye flux through thestratum corneum was apparent in any of the four cells after 12 hrs. ofsonic energy applied as described above.

Example 18

Effects of Sonic Energy without Chemical Enhancers

Perforated Stratum Corneum: Six cells were prepared as described abovein Example 16. The clamps holding the upper and lower chambers of theFranz cells were tightened greater than the extent required to normallyseal the upper compartment from the lower compartment, and to the extentto artificially introduce perforations and “pinholes” into theheat-separated epidermal samples. When dye solution was added to theupper chamber of each cell, there were immediate visual indications ofleakage of dye into the lower chambers through the perforations formedin the stratum corneum. Upon application of sonic energy to cells inwhich the stratum corneum was so perforated with small “pinholes,” arapid increase in the transport of fluid through a pinhole in thestratum corneum was observed. The rate of transport of the indicator dyemolecules was directly related to whether the sonic energy was appliedor not. That is, application of the sonic energy caused an immediate(lag time approximately <0.1 second) pulse of the indicator moleculesthrough the pinholes in the stratum corneum. This pulse of indicatormolecules ceased immediately upon turning off of the sonic energy (ashutoff lag of approximately <0.1 second). The pulse could be repeatedas described.

Example 19 Effects of Sonic Energy and Chemical Enhancers

Two different chemical enhancer formulations were used. ChemicalEnhancer One or CE1 was an admixture of ethanol/glycerol/water/glycerolmonooleate/methyl laurate in a 50/30/15/2.5/2.5 volume ratio. These arecomponents generally regarded as safe, i.e. GRAS, by the FDA for use aspharmaceutical excipients. Chemical Enhancer Two or CE2 is anexperimental formulation shown to be very effective in enhancingtransdermal drug delivery, but generally considered too irritating forlong term transdermal delivery applications. CE2 containedethanol/glycerol/water/lauradone/methyl laurate in the volume ratios50/30/15/2.5/2.5. Lauradone is the lauryl(dodecyl)ester of2-pyrrolidone-5-carboxylic acid (“PCA”) and is also referred to aslauryl PCA.

Six Franz cells were set up as before (Example 16) except that the heatseparated epidermis was installed with the epidermal layer down, i.e.,stratum corneum side facing up. Hydration was established by exposingeach sample to distilled water overnight. To begin the experiment, thedistilled water in the lower chambers was replaced with methylene bluedye solution in all six cells. The upper chambers were filled withdistilled water and the cells were observed for about 30 minutesconfirming no passage of dye to ensure that no pinhole perforations werepresent in any of the cells. When none were found, the distilled waterin the upper chambers was removed from four of the cells. The other twocells served as distilled water controls. The upper chambers of two ofthe experimental cells were then filled with CE1 and the other twoexperimental cells were filled with CE2.

Sonic energy was immediately applied to one of the two CE2 cells. A 25MHz transducer was used with the frequency sweeping every 0.1 secondfrom 10 MHz to 30 MHz at maximum intensity of 0.13 watts/cm². After10-15 minutes of sonic energy applied at a 50% duty cycle, dye flux wasvisually detected. No dye flux was detected in the other five cells.

Sonic energy was then applied to one of the two cells containing CE1 atthe same settings. Dye began to appear in the upper chamber within 5minutes. Thus, sonic energy together with a chemical enhancersignificantly increased the transdermal flux rate of a marker dyethrough the stratum corneum, as well as reduced the lag time.

Example 20 Effects of Sonic Energy and Chemical Enhancers

Formulations of the two chemical enhancers, CE1 and CE2, were preparedminus the glycerin and these new formulations, designated CE1MG andCE2MG, were tested as before. Water was substituted for glycerin so thatthe proportions of the other components remained unchanged. Three cellswere prepared in modified Franz cells with the epidermal side of theheat separated epidermis samples facing toward the upper side of thechambers. These samples were then hydrated in distilled water for 8hours. After the hydration step, the distilled water in the lowerchambers was replaced with either CE1MG or CE2MG and the upper chamberwas filled with the dye solution. Sonic energy was applied to each ofthe three cells sequentially.

Upon application of pulsed, frequency-modulated sonic energy for a totalduration of less than 10 minutes, a significant increase in permeabilityof the stratum corneum samples was observed. The permeability of thestratum corneum was altered relatively uniformly across the area exposedto both the chemical enhancer and sonic energy. No “pinhole”perforations through which the dye could traverse the stratum corneumwere observed. The transdermal flux rate was instantly controllable byturning the sonic energy on or off. Turning the sonic energy offappeared to instantly reduce the transdermal flux rate such that no dyewas visibly being actively transported through the skin sample;presumably the rate was reduced to that of passive diffusion. Turningthe sonic energy on again instantly resumed the high level flux rate.The modulated mode appeared to provide a regular pulsatile increase inthe transdermal flux rate at the modulated rate. When the sonic energywas set to a constant frequency, the maximum increase in transdermalflux rate for this configuration seemed to occur at around 27 MHz.

Having obtained the same results with all three samples, the cells werethen drained of all fluids and flushed with distilled water on bothsides of the stratum corneum. The lower chambers were then immediatelyfilled with distilled water and the upper chambers were refilled withdye solution. The cells were observed for 30 minutes. No holes in thestratum corneum samples were observed and no large amount of dye wasdetected in the lower chambers. A small amount of dye became visible inthe lower chambers, probably due to the dye and enhancer trapped in theskin samples from their previous exposures. After an additional 12hours, the amount of dye detected was still very small.

Example 21 Effects of Sonic Energy and Chemical Enhancers

Perforated Stratum Corneum: Three cells were prepared withheat-separated epidermis samples with the epidermal side facing towardthe upper side of the chamber from the same donor as in Example 16. Thesamples were hydrated for 8 hours and then the distilled water in thelower chambers was replaced with either CE1MG or CE2MG. The upperchambers were then filled with dye solution. Pinhole perforations in thestratum corneum samples permitted dye to leak through the stratumcorneum samples into the underlying enhancer containing chambers. Sonicenergy was applied. Immediately upon application of the sonic energy,the dye molecules were rapidly pushed through the pores. As shown above,the rapid flux of the dye through the pores was directly and immediatelycorrelated with the application of the sonic energy.

Example 22 Effects of Sonic Energy and Chemical Enhancers

A low cost sonic energy transducer, TDK #NB-58S-01 (TDK Corp.), wastested for its capability to enhance transdermal flux rates. The peakresponse of this transducer was determined to be about 5.4 MHz withother local peaks occurring at about 7 MHz, 9 MHz, 12.4 MHz, and 16 MHz.

This TDK transducer was then tested at 5.4 MHz for its ability toenhance transdermal flux rate in conjunction with CE1MG. Three cellswere set up with the epidermal side facing the lower chamber, then theskin samples were hydrated for 8 hrs. The dye solution was placed in thelower chamber. The transducer was placed in the upper chamber immersedin CE1MG. Using swept frequencies from 5.3 to 5.6 MHz as the sonicenergy excitation, significant quantities of dye moved through thestratum corneum and were detected in the collection well of the cell in5 minutes. Local heating occurred, with the transducer reaching atemperature of 48 C. In a control using CE1MG without sonic energy, a 24hour exposure yielded less dye in the collection well than the 5 minuteexposure with sonic energy.

This example demonstrates that a low cost, low frequency sonic energytransducer can strikingly affect transdermal flux rate when used inconjunction with an appropriate chemical enhancer. Although higherfrequency sonic energy will theoretically concentrate more energy in thestratum corneum, when used with a chemical enhancer, the lower frequencymodulated sonic energy can accelerate the transdermal flux rate to makethe technology useful and practical.

Example 23

Demonstration of molecule migration across human skin: Tests with theTDK transducer and CE1MG described above were repeated at about 12.4MHz, one of the highest local resonant peaks for the transducer, with afrequency sweep at a 2 Hz rate from 12.5 to 12.8 MHz and an sonic energydensity less than 0.1 W/cm². The epidermal side of the heat-separatedepidermis was facing down, the dye solution was in the lower chamber,and the enhancer solution and the sonic energy were placed in the upperchamber. Within 5 minutes a significant amount of dye had moved acrossthe stratum corneum into the collection well. Ohmic heating in thetransducer was significantly less than with the same transducer beingdriven at 5.4 MHz, causing an increase in temperature of the chemicalenhancer to only about 33 C.

Even at these low efficiency levels, the results obtained with CE1MG andsonic energy from the TDK transducer were remarkable in the monitoringdirection. FIGS. 3A and 3B of U.S. Pat. No. 5,445,611 show plots of dataobtained from three separate cells with the transdermal flux ratemeasured in the monitoring direction. Even at the 5 minute time point,readily measurable quantities of the dye were present in the chemicalenhancer on the outside of the stratum corneum, indicating transportfrom the epidermal side through the stratum corneum to the “outside”area of the skin sample.

To optimize the use of the sonic energy or the sonic energy/chemicalenhancer approach for collecting and monitoring analytes from the body,means for assaying the amount of analyte of interest are required. Anassay system that takes multiple readings while the unit is in theprocess of withdrawing analytes by sonic energy with or without chemicalenhancers makes it unnecessary to standardize across a broad populationbase and normalize for different skin characteristics and flux rates. Byplotting two or more data points in time as the analyte concentration inthe collection system is increasing, a curve-fitting algorithm can beapplied to determine the parameters describing the curve relatinganalyte withdrawal or flux rate to the point at which equilibrium isreached, thereby establishing the measure of the interval concentration.The general form of this curve is invariant from one individual toanother; only the parameters change. Once these parameters areestablished, solving for the steady state solution (i.e., time equalsinfinity) of this function, i.e., when full equilibrium is established,provides the concentration of the analyte within the body. Thus, thisapproach permits measurements to be made to the desired level ofaccuracy in the same amount of time for all members of a populationregardless of individual variations in skin permeability.

Several existing detection techniques currently exist that are adaptablefor this application. See, D. A. Christensen, in 1648 Proceedings ofFiber Optic, Medical and Fluorescent Sensors and Applications 223-26(1992). One method involves the use of a pair of optical fibers that arepositioned close together in an approximately parallel manner. One ofthe fibers is a source fiber, through which light energy is conducted.The other fiber is a detection fiber connected to a photosensitivediode. When light is conducted through the source fiber, a portion ofthe light energy, the evanescent wave, is present at the surface of thefiber and a portion of this light energy is collected by the detectionfiber. The detection fiber conducts the captured evanescent wave energyto the photosensitive diode which measures it. The fibers are treatedwith a binder to attract and bind the analyte that is to be measured. Asanalyte molecules bind to the surface (such as the analyte glucosebinding to immobilized lectins such as concanavalin A, or to immobilizedanti-glucose antibodies) the amount of evanescent wave coupling betweenthe two fibers is changed and the amount of energy captured by thedetection fiber and measured by the diode is changed as well. Severalmeasurements of detected evanescent wave energy over short periods oftime support a rapid determination of the parameters describing theequilibrium curve, thus making possible calculation of the concentrationof the analyte within the body. The experimental results showingmeasurable flux within 5 minutes (FIGS. 3A and 3B of U.S. Pat. No.5,445,611) with this system suggest sufficient data for an accuratefinal reading are collected within 5 minutes.

In its most basic embodiment, a device that can be utilized for theapplication of sonic energy and collection of analyte comprises anabsorbent pad, either of natural or synthetic material, which serves asa reservoir for the chemical enhancer, if used, and for receiving theanalyte from the skin surface. The pad or reservoir is held in place,either passively or aided by appropriate fastening means, such as astrap or adhesive tape, on the selected area of skin surface.

An sonic energy transducer is positioned such that the pad or reservoiris between the skin surface and the transducer, and held in place byappropriate means. A power supply is coupled to the transducer andactivated by switch means or any other suitable mechanism. Thetransducer is activated to deliver sonic energy modulated in frequency,phase or intensity, as desired, to deliver the chemical enhancer, ifused, from the reservoir through the skin surface followed by collectionof the analyte from the skin surface into the reservoir. After thedesired fixed or variable time period, the transducer is deactivated.The pad or reservoir, now containing the analyte of interest, can beremoved to quantitate the analyte, for example, by a laboratoryutilizing any number of conventional chemical analyses, or by a portabledevice. Alternately, the mechanism for quantitating the analyte can bebuild into the device used for collection of the analyte, either as anintegral portion of the device or as an attachment. Devices formonitoring an analyte are described in U.S. Pat. No. 5,458,140, which isincorporated herein by reference.

Example 24

An alternate method for detection of an analyte, such as glucose,following the sample collection through the porated skin surface asdescribed above, can be achieved through the use of enzymatic means.Several enzymatic methods exist for the measurement of glucose in abiological sample. One method involves oxidizing glucose in the samplewith glucose oxidase to generate gluconolactone and hydrogen peroxide.In the presence of a colorless chromogen, the hydrogen peroxide is thenconverted by peroxidase to water and a colored product.

Glucose Oxidase

Glucose Gluconolactone+H₂O₂

2H₂0₂+chromogen H₂O+colored product

The intensity of the colored product will be proportional to the amountof glucose in the fluid. This color can be determined through the use ofconventional absorbance or reflectance methods. By calibration withknown concentrations of glucose, the amount of color can be used todetermine the concentration of glucose in the collected analyte. Bytesting to determine the relationship, one can calculate theconcentration of glucose in the blood of the subject. This informationcan then be used in the same way that the information obtained from ablood glucose test from a finger puncture is used. Results can beavailable within five to ten minutes.

Example 25

Any system using a visual display or readout of glucose concentrationwill indicate to a diagnostician or patient the need for administrationof insulin or other appropriate medication. In critical care or othersituations where constant monitoring is desired and corrective actionneeds to be taken almost concurrently, the display may be connected withappropriate signal means which triggers the administration of insulin orother medication in an appropriate manner. For example, there areinsulin pumps which are implanted into the peritoneum or other bodycavity which can be activated in response to external or internalstimuli. Alternatively, utilizing the enhanced transdermal flux ratespossible with micro-poration of the stratum corneum and other techniquesdescribed in this invention, an insulin delivery system could beimplemented transdermally, with control of the flux rates modulated bythe signal from the glucose sensing system. In this manner a completebiomedical control system can be available which not only monitorsand/or diagnoses a medical need but simultaneously provides correctiveaction.

Biomedical control systems of a similar nature could be provided inother situations such as maintaining correct electrolyte balances oradministering analgesics in response to a measured analyte parametersuch as prostaglandins.

Example 26

Similar to audible sound, sonic waves can undergo reflection,refraction, and absorption when they encounter another medium withdissimilar properties [D. Bommannan et al., 9 Pharm. Res. 559 (1992)].Reflectors or lenses may be used to focus or otherwise control thedistribution of sonic energy in a tissue of interest. For many locationson the human body, a fold of flesh can be found to support this system.For example, an earlobe is a convenient location which would allow useof a reflector or lens to assist in exerting directional control (e.g.,“pushing” of analytes or permeants through the porated stratum corneum)similar to what is realized by changing sonic frequency and intensity.

Example 27

Multiple sonic energy transducers may be used to selectively direct thedirection of transdermal flux through porated stratum corneum eitherinto the body or from the body. A fold of skin such as an earlobe allowtransducers to be located on either side of the fold. The transducersmay be energized selectively or in a phased fashion to enhancetransdermal flux in the desired direction. An array of transducers or anacoustic circuit may be constructed to use phased array concepts,similar to those developed for radar and microwave communicationssystems, to direct and focus the sonic energy into the area of interest.

Example 28

In this example, the procedure of Example 19 is followed with theexception that the heat-separated epidermis samples are first treatedwith an excimer laser (e.g. model EMG/200 of Lambda Physik; 193 nmwavelength, 14 ns pulse width) to ablate the stratum corneum accordingto the procedure described in U.S. Pat. No. 4,775,361, herebyincorporated by reference.

Example 29

In this example, the procedure of Example 19 is followed with theexception that the heat-separated epidermis samples are first treatedwith 1,1′-diethyl-4,4′-carbocyanine iodide (Aldrich, _(max)=703 nm) andthen a total of 70 mJ/cm²/50 ms is delivered to the dye-treated samplewith a Model TOLD9150 diode laser (Toshiba America Electronic, 30 mW at690 nm) to ablate the stratum corneum.

Example 30

In this example, the procedure of Example 29 is followed with theexception that the dye is indocyanine green (Sigma cat. no. I-2633;_(max)=775 nm) and the laser is a model Diolite 800-50 (LiCONiX, 50 mWat 780 nm).

Example 31

In this example, the procedure of Example 29 is followed with theexception that the dye is methylene blue and the laser is a modelSDL-8630 (SDL Inc.; 500 mW at 670 nm).

Example 32

In this example, the procedure of Example 29 is followed with theexception that the dye is contained in a solution comprising apermeation enhancer, e.g. CE1.

Example 33

In this example, the procedure of Example 29 is followed with theexception that the dye and enhancer-containing solution are delivered tothe stratum corneum with the aid of exposure to ultrasound.

Example 34

In this example, the procedure of Example 31 is followed with theexception that the pulsed light source is a short arc lamp emitting overthe broad range of 400 to 1100 nm but having a bandpass filter placed inthe system to limit the output to the wavelength region of about 650 to700 nm.

Example 35

In this example, the procedure of Example 19 is followed with theexception that the heat-separated epidermis samples are first puncturedwith a microlancet (Becton Dickinson) calibrated to produce a microporein the stratum corneum without reaching the underlying tissue.

Example 36

In this example, the procedure of Example 19 is followed with theexception that the heat-separated epidermis samples are first treatedwith focused sonic energy in the range of 70-480 mJ/cm²/50 ms to ablatethe stratum corneum.

Example 37

In this example, the procedure of Example 19 is followed with theexception that the stratum corneum is first punctured hydraulically witha high pressure jet of fluid to form a micropore of up to about 100 μmdiameter.

Example 38

In this example, the procedure of Example 19 is followed with theexception that the stratum corneum is first punctured with short pulsesof electricity to form a micropore of up to about 100 μm diameter.

Example 39 Acoustic Streaming

A new mechanism and application of sonic energy in the delivering oftherapeutic substances into the body and/or harvesting fluids fromwithin the body into an external reservoir through micro-porationsformed in the biological membrane will now be described. An additionalaspect of this invention is the utilization of sonic energy to create anacoustic streaming effect on the fluids flowing around and between theintact cells in the viable tissues beneath the outer layer of anorganism, such as the epidermis and dermis of the human skin. Acousticstreaming is a well documented mode by which sonic energy can interactwith a fluid medium. Nyborg, Physical Acoustics Principles and Methods,p. 265-331, Vol II-Part B, Academic Press, 1965. The first theoreticalanalysis of acoustic streaming phenomenon was given by Rayleigh (1884,1945). In an extensive treatment of the subject, Longuet-Higgins(1953-1960) has given a result applicable to two dimensional flow thatresults in the near vicinity of any vibrating cylindrical surface. Athree dimensional approximation for an arbitrary surface was developedby Nyborg (1958). As described by Fairbanks et al., 1975 UltrasonicsSymposium Proceedings, IEEE Cat. #75, CHO 994-4SU, sonic energy, and theacoustic streaming phenomenon can be of great utility in acceleratingthe flux of a fluid through a porous medium, showing measurableincreases in the flux rates by up to 50 times that possible passively orwith only pressure gradients being applied.

All previous transdermal delivery or extraction efforts utilizingultrasound have focused on methods of interaction between the sonicenergy and the skin tissues designed to permeabilize the stratum corneumlayer. The exact mode of interaction involved has been hypothesized tobe due exclusively to the local elevation of the temperature in the SClayer, and the resultant melting of the lipid domains in theintercellular spaces between the corneocytes. Srinivasan et al. Otherresearchers have suggested that micro-cavitations and or shearing of thestructures in the stratum corneum opens up channels through which fluidsmay flow more readily. In general, the design of the sonic systems forthe enhancement of transdermal flux rates has been based on the earlyrealization that the application of an existing therapeutic ultrasoundunit designed to produce a “deep-heating” effect on the subject, whenused in conjunction with a topical application of a gelled or liquidpreparation containing the drug to be delivered into the body, couldproduce a quantifiable increase in the flux rate of the drug into thebody. In the context of the method taught herein to create micropores inthis biological membrane, the use of sonic energy may now be thought ofin a totally new and different sense than the classically definedconcepts of sonophoresis.

Based on the experimental discovery mentioned in U.S. Pat. Nos.5,458,440 and 5,445,611 that when a small hole existed or was created inthe stratum corneum (SC) in the Franz cells used in the in vitrostudies, that the application of an appropriately driven ultrasonictransducer to the fluid reservoir on either side of the porated SCsample, an “acoustic streaming” event could be generated wherein largeflux rates of fluid where capable of being pumped through this poratedmembrane.

With the method taught herein to create the controlled micro-porationsin the biological membrane in the organism, the application of the fluidstreaming mode of sonic/fluid interaction to the induction of fluid intoor out of the organism may now be practically explored. For example,clinical studies have shown that by making a series of four 80 μmdiameter micropores in a 400 μm square, and then applying a mild (10 to12 inches of Hg) suction to this area, an average of about 1 μl ofinterstitial fluid can be induced to leave the body for externalcollection in an external chamber. By adding a small, low power sonictransducer to this system, configured such that it actively generatesinwardly converging concentric circular pressure waves in the 2 to 6 mmof tissue surrounding the poration site, it has been demonstrated thatthis ISF flux rate can be increased by 50%.

By relieving ourselves of the desire to create some form of directabsorption of sonic energy in the skin tissues (as required to generateheating), frequencies of sonic energy can be determined for which theskin tissues are virtually transparent, that is at the very lowfrequency region of 1 kHz to 500 KHz. Even at some of the lowestfrequencies tested, significant acoustic streaming effects could beobserved by using a micro-scope to watch an in vivo test wherein thesubject's skin was micro-porated and ISF was induced to exit the body anpool on the surface of the skin. Energizing the sonic transducer showeddramatic visual indications of the amount of acoustic streaming as smallpieces of particulate matter were carried along with the ISF as itswirled about. Typical magnitude of motion exhibited can be described asfollows: for a 3 mm diameter circular pool of ISF on the surface of theskin, a single visual particle could be seen to be completing roughly 3complete orbits per second. This equates to a linear fluid velocity ofmore than 2.5 mm/second. All of this action was demonstrated with sonicpower levels into the tissues of less than 100 mW/cm2.

While one can easily view the top surface of the skin, and the fluidicactivity thereon, assessing what is taking place dynamically within theskin tissue layers in response to the coupling into these tissues ofsonic energy is much more difficult. One can assume, that if such largefluid velocities (e.g. >2.5 mm/S) may be so easily induced on thesurface, then some noticeable increase in the fluid flow in theintercellular channels present in the viable dermal tissues could alsobe realized in response to this sonic energy input. Currently, anincrease in harvested ISF through a given set of microporations when alow frequency sonic energy was applied to the area in a circlesurrounding the poration sites has been quantified. In this experiment,an ISF harvesting technique based solely on a mild suction (10 to 12inches of HG) was alternated with using the exact same apparatus, butwith the sonic transducer engaged. Over a series of 10 two-minuteharvesting periods, five with mere suction and five with both suctionand sonic energy active, it was observed that by activating the sonicsource roughly 50% more ISF was collectable in the same time period.These data are shown in FIG. 30. This increase in ISF flux rate wasrealized with no reported increase in sensation from the test subjectdue to the sonic energy. The apparatus used for this experiment isillustrated in FIGS. 31-33. The transducer assembly in FIGS. 31-33 iscomprised of a thick walled cylinder of piezo-electric material, with aninternal diameter of roughly 8 mm and a wall thickness of 4 mm. Thecylinder has been polarized such that when an electrical field isapplied across the metallized surfaces of the outer diameter and innerdiameter, the thickness of the wall of the cylinder expands or contractsin response to the field polarity. In practice, this configurationresults in a device which rapidly squeezes the tissue which has beensuctioned into the central hole, causing an inward radial acousticstreaming effect on those fluids present in these tissues. This inwardacoustic streaming is responsible for bringing more ISF to the locationof the micro-porations in the center of the hole, where it can leave thebody for external collection.

A similar device shown in FIG. 34A-B was built and tested and producedsimilar initial results. In the FIG. 34A-B version, an ultrasonictransducer built by Zevex, Inc. Salt Lake City, Utah, was modified byhaving a spatulate extension added to the sonic horn. A 4 mm hole wasplaced in the 0.5 mm thick spatulate end of this extension. Whenactivated, the principle motion is longitudinal along the length of thespatula, resulting in essentially a rapid back and forth motion. Thephysical perturbation of the metallic spatula caused by the placement ofthe 4 mm hole, results in a very active, but chaotic, large displacementbehavior at this point. In use, the skin of the subject was suctioned upinto this hole, and the sonic energy was then conducted into the skin ina fashion similar to that illustrated in FIG. 33.

The novel aspect of this new application of ultrasound lies in thefollowing basic areas:

1. The function of the sonic energy is no longer needed to be focused onpermeabilizing the SC barrier membrane as taught by Langer, Kost,Bommannan and others.

2. A much lower frequency system can be utilized which has very littleabsorption in the skin tissues, yet can still create the fluidicstreaming phenomenon desired within the intercellular passagewaysbetween the epidermal cells which contain the interstitial fluid.

3. The mode of interaction with the tissues and fluids therein, is theso-called “streaming” mode, recognized in the sonic literature as aunique and different mode than the classical vibrational interactionscapable of shearing cell membranes and accelerating the passivediffusion process.

By optimizing the geometric configuration, frequency, power andmodulations applied to the sonic transducer, it has been shown thatsignificant increases in the fluid flux through the porated skin sitescan be achieved. The optimization of these parameters is designed toexploit the non-linearities governing the fluid flow relationships inthis microscopically scaled environment. Using frequencies under 200kHz, large fluidic effects can be observed, without any detectableheating or other negative tissue interactions. The sonic power levelsrequired to produce these measurable effects are very low, with averagepower levels typically under 100 milliwatts/cm2.

Therefore, the above examples are but representative of systems whichmay be employed in the utilization of sonic energy or sonic energy andchemical enhancers in the collection and quantification of analytes fordiagnostic purposes and for the transmembrane delivery of permeants. Theinvention is directed to the discovery that the poration of thebiological membrane followed by the proper use of sonic energy,particularly when accompanied with the use of chemical enhancers,enables the noninvasive or minimally invasive transmembranedetermination of analytes or delivery of permeants. However, theinvention is not limited only to the specific illustrations. There arenumerous poration techniques and enhancer systems, some of which mayfunction better than another, for detection and withdrawn of certainanalytes or delivery of permeants through the stratum corneum. However,within the guidelines presented herein, a certain amount ofexperimentation to obtain optimal poration, enhancers, or optimal time,intensity and frequency of applied sonic energy, as well as modulationof frequency, amplitude and phase of applied sonic energy can be readilycarried out by those skilled in the art. Therefore, the invention islimited in scope only by the following claims and functional equivalentsthereof.

Further Advancements and Improvements

Advancements and improvements to the microporation techniques have beenmade, particularly suitable for, though not limited to, deliveryapplications. One advancement is to porate, using any one of theaforementioned microporation techniques, to a selected depth into orthrough biological membranes, including the skin, the mucous membrane,or plant outer layer, particularly for delivery of a drug or bioactiveagent into the body. Another advancement is to deliver bioactive agentsinto the organism through micropores formed in the biological membrane.Still another advancement is to apply permeation enhancement measuresbefore, during, or after microporation, so as to increase thepermeability of layers within the microporated skin or mucosa whendelivering substances, such as drugs or bioactive agents, thereinto ortherethrough.

The micropore formed in the biological membrane may extend to a selecteddepth. A micropore extending into the epidermis may penetrate only thestratum corneum or selected depths into the viable cell layer orunderlying connective tissue layer. Similarly, if formed in the mucousmembrane, the micropore may penetrate only the superficial part of theepithelial layer or selected depths into the epithelial lining orunderlying lamina propria and into tissue beneath. The micropore depthin either case can extend through the entire depth of the biologicalmembrane.

As an example for microporating to a selected depth, if one utilizes aheat probe which can continue to deliver sufficient energy into orthrough the fully hydrated viable cell layers beneath the stratumcorneum, the poration process can continue into the body to selecteddepths, penetrating through the epidermis, the dermis, and into orthrough the subcutaneous layers below if desired. The concern when asystem is designed to create a micropore extending some distance into orthrough the viable tissues in the epidermis or dermis, or the epitheliallining or lamina propria, is how to minimize damage to the adjacenttissue and the sensation to the subject during the poration process.

Experimentally, we have shown that if the heat probe used is a solid,electrically or optically heated element, with the active heated probetip physically defined to be no more than a few hundred microns acrossand protruding up to a few millimeters from the supporting base, that asingle pulse, or multiple pulses of current can deliver enough thermalenergy into the tissue to allow the ablation,to penetrate as deep as thephysical design allows, that is, until the support base limits theextent of the penetration into or through the tissue. If the electricaland thermal properties of said heat probe, when it is in contact withthe tissues, allow the energy pulse to modulate the temperature of saidprobe rapidly enough, this type of deep tissue poration can beaccomplished with essentially no pain to the subject. Experiments haveshown that if the required amount of thermal energy is delivered to theprobe within less than roughly 20 milliseconds (20-50 msec), that theprocedure is painless. Conversely, if the energy pulse must be extendedbeyond roughly 20 milliseconds (20-50 msec), the sensation to thesubject increases rapidly and non-linearly as the pulse width isextended.

Similarly, an electrically heated probe design which supports this typeof selected deep poration can be built by bending a 50 to 150 microndiameter tungsten wire into a sharp kink, forming a close to 180 degreebend with a minimal internal radius at this point. This miniature ‘V’shaped piece of wire can then be mounted such that this ‘V’ extends somedistance out from a support piece which has copper electrodes depositedupon it. The distance to which the wire extends out from the supportwill define the maximum penetration distance into the tissue when thewire is heated. Each end of the tungsten ‘V’ will be attached to one ofthe electrodes on the support carrier which in turn can be connected tothe current pulsing circuit. When the current is delivered to the wirein an appropriately controlled fashion, the wire will rapidly heat up tothe desired temperature to effect the thermal ablation process in asingle pulse or in multiple pulses of current. By monitoring the dynamicimpedance of the probe and knowing the coefficient of resistance versustemperature of the tungsten element, closed loop control of thetemperature of the contact point can easily be established. Also, bydynamically, monitoring the impedance through the body from the contactpoint of the probe and a second electrode placed some distance away, thedepth of the pore can be determined based on the different impedanceproperties of the tissue as one penetrates deeper into the body. Oncethe impedance properties of a selected tissue of a selected organismhave been routinely determined, this parameter can be used to determinethe pore depth and can be used in a control system to control poredepth.

Likewise, one embodiment of an optically heated probe design whichsupports this type of selected depth poration can be built by taking anoptical fiber and placing on one end a tip comprised of a solid cap orcoating. A light source such as a laser diode will be coupled into theother end of the fiber. The side of tip facing the fiber must have ahigh enough absorption coefficient over the range of wavelengths emittedby the light source that when the photons reach the end of the fiber andstrike this face, some of them will be absorbed and subsequently causethe tip to heat up. The specific design of this tip, fiber and sourceassembly may vary widely, however fibers with gross diameters of 50 to1000 microns across are common place items today and sources emitting upto thousands of watts of optical energy are similarly common place. Thetip forming the actual heat probe can be fabricated from a high meltingpoint material, such as tungsten and attached to the fiber by machiningit to allow the insertion of the fiber into a cylindrical bore at thefiber end. If the distal end of the tip has been fabricated to limit thethermal diffusion away from this tip and back up the supporting cylinderattaching the tip to the fiber within the time frame of the opticalpulse widths used, the photons incident upon this tip will elevate thetemperature rapidly on both the fiber side and the contact side which isplaced against the tissues surface. The positioning of the fiber/tipassembly onto the tissue surface, can be accomplished with a simplemechanism designed to hold the tip against the surface under some springtension such that as the tissue beneath it is ablated, the tip itselfwill advance into the tissue. This allows the thermal ablation processto continue into or through the tissue as far as one desires. Anadditional feature of this optically heated probe design is that bymonitoring the black body radiated energy from the heated tip that iscollected by the fiber, a very simple closed loop control of the tiptemperature can be effected. Also, as described earlier, by dynamicallymonitoring the impedance through the body from the contact point of theprobe and a second electrode placed some distance away, the depth of thepore can be estimated based on the different impedance properties of thetissue as one penetrates deeper into the body. The relationship betweenpulse width and sensation for this design is essentially the same as forthe electrically heated probe described earlier.

For example, some vaccine applications are known to be most effective ifdelivered into the dermal layer so as to be in proximity to theLangerhan's or dendritic cells or other cells important for this immuneresponse. This would imply a poration depth designed to pass through theepidermis, which in most cases would be roughly 180 microns to 250microns deep.

As another example, when delivering some proteins and peptides, it isdesirable to minimize the immune response to the permeant at the site ofthe administration and at the same time bypass the protease active zonesin the skin tissues. In this case an even deeper pore may be desired,going as deep as 300 microns into the skin.

Alternatively, it may be desirable to leave a minimally thick layer ofintact stratum corneum to minimize rapid initial uptake of a permeantand to provide some retention of the stratum corneum's barrier functionto provide for a controlled release over a longer period of time.

An additional feature of this invention is the large increase inefficiency which can be gained by combining the poration of the layersof the biological membrane with other permeation enhancement techniqueswhich can now be optimized to function on the various barriers to effectdelivery of the desired compound into the internal spaces as necessaryfor bio-effectivity. In particular, if one is delivering a nucleic acidcompound either naked, fragmented, encapsulated or coupled to anotheragent, it is often desired to get the nucleic acid into the living cellswithout killing the cell to allow the desired uptake and subsequentperformance of the therapy. The application of electroporation,iontophoresis, magnetic fields and thermal and sonic energy can causeopenings to form, temporarily, in the cell membranes and other internaltissues. Because we have shown how to breach the stratum corneum orepithelial layer of the mucosal membrane or the outer layer of a plant,and if desired the epidermis and dermis or deeper into a plant,electroporation, iontophoresis, magnetic fields and thermal and sonicenergy can now be used with parameters that can be tailored to actselectively on these underlying tissue barriers and permeabilize thecell, capillary or other membranes within the targeted tissue.Electroporation, iontophoresis, magnetic fields, and thermal and sonicenergy were previously inapplicable for this use.

In the case of electroporation, where pulses exceeding 50 to 150 voltsare routinely used to electroporate the stratum corneum or outer layerof the mucosal membrane or outer layer of a plant, in the environment wepresent, pulses of only a few volts or less are sufficient toelectroporate the cell, capillary or other membranes within the targetedtissue. This is principally due to the dramatic reduction in the numberof insulating layers present between the electrodes once the skin,mucosal layer, or outer layer of a plant has been opened.

Similarly, iontophoresis can be shown to be effective to modulate theflux of a fluid media containing the nucleic acid through the microporeswith very small amounts of current due to the dramatic reduction in thephysical impedance to fluid flow through these porated layers.

In the case of sonic energy, whereas classically sonic energy has beenused to accelerate the permeation of the stratum corneum or mucosallayer, by eliminating this barrier, sonic energy can now be used topermeabilize the cell, capillary or other membranes within the targetedtissue. As in the cases of electroporation and iontophoresis, we havedemonstrated that the sonic energy levels needed to effect a notableimprovement in the transmembrane flux of a substance are much lower thanwhen skin or mucosal layers are left intact. Other permeationenhancement measures involve changing the osmotic pressure or physicalpressure at the microporated site, for example applying a mild pneumaticpressure to the permeant reservoir to force a particular fluid flow intothe organism through the micropores

The mode of operation of all of these active methods, electroporation,iontophoresis, magnetic or thermal or sonic energy, when applied solelyor in combination, after the poration of the skin or mucosal layer orthe outer layer of a plant has been effected, has the advantage of beingable to use parameters typically used in in vitro applications wheresingle cell membranes are opened up for the delivery of a substance.Examples of these parameters are well known in the literature. Forexample, Sambvrook et al., Molecular Cloning: A Laboratory Manual, 2dEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.

The micropores produced in the biological membrane by the methods of thepresent invention allow high flux rates of large (as well as small)molecular weight therapeutic compounds to be delivered transdermally ortransmucosally or transmembrane. In addition, these non-traumaticmicroscopic openings into the body allow access to various analyteswithin the body, which can be assayed to determine their internalconcentrations.

Delivery of Bioactive Agents

Still another advancement of the present invention involves the use ofporation of the biological membrane for the delivery of a bioactiveagent, e.g., polypeptides, including proteins and peptides (e.g.,insulin); releasing factors; including LHRH; carbohydrates (e.g.,heparin); nucleic acids; vaccines; and pharmacologically active agentssuch as antiinfectives such as antibiotics and antiviral agents;analgesics and analgesic combinations; anorexics; antihelminthics;antiarthritics; antiasthmatic agents; anticonvulsants; antidepressants;antidiabetic agents; antidiarrheals; antihistamines; antiinflammatoryagents; antimigraine preparations; antinauseants; antineoplastics;antiparkinsonism drugs; antipruritics; antipsychotics; antipyretics;antispasmodics; anticholinergics; sympathomimetics; xanthinederivatives; cardiovascular preparations including potassium and calciumchannel blockers, beta-blockers, alpha-blockers, and antiarrhythmics;antihypertensives; diuretics and antidiuretics; vasodilators includinggeneral coronary, peripheral and cerebral; central nervous systemstimulants; vasoconstrictors; cough and cold preparations, includingdecongestants; hormones such as estradiol, testosterone, progesteroneand other steroids and derivatives and analogs, includingcorticosteroids; hypnotics; immunosuppressives; muscle relaxants;parasympatholytics; psychostimulants; sedatives; and tranquilizers. Bythe method of the present invention, both ionized and nonionized drugsmay be delivered, as can drugs of either high, medium or low molecularweight.

Delivery of DNA and/or RNA can be used to achieve expression of apolypeptide, stimulate an immune response, or to inhibit expression of apolypeptide through the use of an “antisense” nucleic acid, especiallyan antisense RNA. The term “polypeptide” is used herein without anyparticular intended size limitation, unless a particular size isotherwise stated, and includes peptides of any length includingproteins. Typical of polypeptides that can be expressed are thoseselected from the group consisting of oxytocin, vasopressin,adrenocorticotrophic hormone, epidermal growth factor, prolactin,luteinizing hormone releasing hormone, growth hormone, growth hormonereleasing factor, insulin-like growth factors, insulin, erythropoietin,obesity protein such as leptin, somatostatin, glucagon, glucagon-likeinsulinotropic factors, parathyroid hormone, interferon, gastrin,interleukin-2 and other interleukins and lymphokines, tetragastrin,pentagastrin, urogastroine, secretin, calcitonin, enkephalins,endorphins, angiotensins, renin, bradykinin, bacitracins, polymixins,colistins, tyrocidin, gramicidines, and synthetic analogues,modifications and pharmacologically active fragments thereof, monoclonalantibodies and vaccines. This group is not to be considered limiting;the only limitation to the peptide or protein drug that may be expressedis one of functionality. Delivery of DNA and/or RNA is useful in genetherapy, vaccination, and any therapeutic situation in which a nucleicacid or a polypeptide should be administered in vivo. E.g., U.S. Pat.No. 5,580,859, hereby incorporated by reference.

One illustrative embodiment of the invention is a method for obtaininglong term administration of a polypeptide comprising porating thebiological membrane and then delivering a DNA encoding the polypeptidethrough the pores in the biological membrane, whereby cells of thetissue take up the DNA and produce the polypeptide for at least onemonth, and more preferably at least 6 months. Another illustrativeembodiment of the invention is a method for obtaining transitoryexpression of a polypeptide comprising porating the biological membraneand then delivering an RNA or DNA encoding the polypeptide through thepores of the biological membrane, whereby cells of the tissue (e.g., theskin, mucous membrane, capillaries, or underlying tissue) take up theRNA or DNA and produce the polypeptide for less than about 20 days,usually less than about 10 days, and often less than about 3-5 days. Thecells which take up the RNA or DNA could include the cells of thebiological membrane, the underlying tissue or other target tissuereached by way of the capillaries.

The DNA and/or RNA can be naked nucleic acid optionally in a carrier orvehicle, and/or can be contained within microspheres, liposomes and/orassociated with transfection-facilitating proteins, microparticles,lipid complexes, viral particles, charged or neutral lipids,carbohydrates, calcium phosphate or other precipitating agents, and/orother substances for stabilizing the nucleic acid. The nucleic acid canbe contained in a viral vector that either integrates into thechromosome or is nonintegrating, in a plasmid, or as a nakedpolynucleotide. The nucleic acid can encode a polypeptide, oralternatively, can code for an antisense RNA, for example for inhibitingtranslation of a selected polypeptide in a cell. When the nucleic acidis DNA, it can be a DNA sequence that is itself non-replicating, but isinserted into a plasmid wherein the plasmid further comprises areplicator. The DNA may also contain a transcriptional promoter, such asthe CMV IEP promoter, which is functional in humans. The DNA can alsoencode a polymerase for transcribing the DNA. In one preferredembodiment, the DNA codes for both a polypeptide and a polymerase fortranscribing the DNA. The DNA can be delivered together with thepolymerase or with mRNA coding therefor, which mRNA is translated in thecell. In this embodiment, the DNA is preferably a plasmid, and thepolymerase is preferably a phage polymerase, such as the T7 polymerase,wherein the T7 polymerase gene should include a T7 promoter.

The method can be used to treat a disease associated with a deficiencyor absence or mutation of a specific polypeptide. In accordance withanother aspect of the invention, the method provides for immunizing anindividual, wherein such individual can be a human or an animal,comprising delivering a DNA and/or RNA to the individual wherein the DNAand/or RNA codes for an immunogenic translation product that elicits animmune response against the immunogen. The method can be used to elicita humoral immune response, a cellular immune response, or a mixturethereof.

Example 40

This illustrative example shows the preparation and delivery of an mRNA.

In general, it should be apparent that, in practicing the invention, asuitable plasmid for in vitro transcription of mRNA can be readilyconstructed by those of ordinary skill in the art with a virtuallyunlimited number of cDNAs. Such plasmids can advantageously comprise apromoter for a selected RNA polymerase, followed by a 5′ untranslatedregion, a 3′ untranslated region, and a template for a polyadenylatetract. There should be a unique restriction site between these 5′ and 3′untranslated regions to facilitate the insertion of any selected cDNAinto the plasmid. Then, after cloning the plasmid containing theselected gene, the plasmid is linearized by digestion in thepolyadenylation region and is transcribed in vitro to form mRNAtranscripts. These transcripts are preferably provided with a 5′ cap.Alternatively, a 5′ untranslated sequence such as EMC can be used, whichdoes not require a 5′ cap.

The readily available SP6 cloning vector, pSP64T, provides 5′ and 3′flanking regions from the Xenopus-globin gene, an efficiently translatedmRNA. Any cDNA containing an initiation codon can be introduced intothis plasmid, and mRNA can be prepared from the resulting template DNA.This particular plasmid can be digested with BglII to insert anyselected cDNA coding for a polypeptide of interest. Although goodresults can be obtained with pSP64T when linearized and then transcribedwith SP6 RNA polymerase, it is preferable to use the Xenopus-globinflanking sequences of pSP64T with the phage T7 RNA polymerase. This isaccomplished by purifying an approximately 150 by HindIII/EcoRI fragmentfrom pSP64T and inserting it into a linearized approximately 2.9 kbHindIII/EcoRI fragment of pIBI131 (commercially available fromInternational Biotechnologies, Inc., New Haven, Conn.) with T4 ligase.The resulting plasmid, pXBG, is adapted to receive any gene of interestat a unique BglII site situated between the two Xenopus-globin sequencesand for transcription of the selected gene with T7 polymerase.

A convenient marker gene for demonstrating in vivo expression ofexogenous polynucleotides is chloramphenicol acetyltransferase, CAT. TheCAT gene from the small BamHI/HindIII fragment of pSV2-CAT (ATCC No.37155) and the BglII-digested pXBG are both incubated with the Klenowfragment of E. coli DNA polymerase to generate blunt ends, and then areligated with T4 DNA ligase to form pSP-CAT. This plasmid is thendigested with PstI and HindIII and the small fragment, comprising theCAT gene between the 5′ and 3′-globin flanking sequences of pSP64T. TheT7 promoter-containing plasmid pIBI131 is also digested with PstI andHindIII, and the long fragment is purified. This fragment is thenligated to the CAT gene containing fragment with T4 DNA ligase to formthe plasmid pT7CAT-An.

The pT7CAT-An plasmid DNA is purified according to methods well known inthe art, e.g. U.S. Pat. No. 5,580,859. The resulting purified plasmidDNA is then linearized downstream of the polyadenylate region with anexcess of PstI, and the resulting linearized DNA is then purified andtranscribed in vitro according to the method of Example 5 of U.S. Pat.No. 5,580,859. The resulting mRNA is then purified according to themethod of Example 5 of U.S. Pat. No. 5,580,859, which is sufficientlypure for delivery according the present invention.

The purified mRNA is delivered by porating a selected site on anindividual according to the microporation procedures with selected poredepth which optimizes bioactivity and delivering an effective amount ofmRNA to such site such that the mRNA passes through the skin or mucousmembrane into the underlying tissue, where the mRNA is taken up by thecells. This delivery through the porated stratum corneum or mucousmembrane can be aided with sonic energy and/or use sonic energyaccording to the procedure of Example 15 and/or with electroporation toenhance cellular uptake, and/or with a pressure differential forinducing flux through the pores in the skin or mucous membrane.Moreover, delivery can be aided by placing the mRNA is a carriersolution, such as a positively charged lipid complex or liposome, forenhancing the diffusion of the mRNA through the pores into the body orfor facilitating uptake of the mRNA into cells.

Example 41

This example shows immunization of an individual with mRNA encoding thegp120 protein of HIV. The mRNA is prepared according to the procedure ofExample 40 except the gene for gp120 (pIIIenv3-1 from the AIDS Researchand Reagent Program, National Institute of Allergy and InfectiousDisease, Rockville, Md.) is inserted into the plasmid pXBG of Example40. The mRNA containing the gp120 gene is delivered according to theprocedure of Example 40.

Example 42

This example shows immunization of an individual with DNA encoding thegp120 protein of HIV. The gp120 gene is inserted into a recombinantadenovirus according to the procedure of P. Muzzin et al., Correction ofObesity and Diabetes in Genetically Obese Mice by Leptin Gene Therapy,93 Proc. Nat'l Acad. Sci. USA 14804-14808 (1996); G. Chen et al.,Disappearance of Body Fat in Normal Rats Induced by Adenovirus-mediatedLeptin Gene Therapy, 93 Proc. Nat'l Acad. Sci. USA 14795-99 (1996),hereby incorporated by reference. The resulting DNA is deliveredaccording to the procedure of Example 41.

Example 43

In this example, the procedure of Example 42 is followed except that DNAencoding glycoprotein D of HSV-2 is substituted for the DNA encodinggp120 protein and additionally is combined with an effective amount ofthe glycoprotein D.

Example 44

In this example, a nucleic acid encoding the obesity protein leptin,such as a human leptin or a rat leptin cDNA, C. Guoxun et al.,Disappearance of Body Fat in Normal Rats Induced by Adenovirus-mediatedLeptin, 93 Proc. Nat'l Acad. Sci. USA 14795-99 (1996), or a mouse leptincDNA, P. Muzzin et al., Correction of Obesity and Diabetes inGenetically Obese Mice by Leptin Gene Therapy, 93 Proc. Nat'l Acad. Sci.USA 14804-14808 (1996), both of which are hereby incorporated byreference, is delivered in an appropriate plasmid vector. The mammalianexpression vector, pEUK-C1 (Clontech, Palo Alto, Calif.) is designed fortransient expression of cloned genes. This vector is a 4.9 kb plasmidcomprising a pBR322 origin of replication and an ampicillin resistancemarker for propagation in bacteria, and also comprising the SV40 originof replication, SV40 late promoter, and SV40 late polyadenylation signalfor replication and expression of a selected gene in a mammalian cell.Located between the SV40 late promoter and SV40 late polyadenylationsignal is a multiple cloning site (MCS) of unique XhoI, XbaI, SmaI,SacI, and BamHI restriction sites. DNA fragments cloned into the MCS aretranscribed into RNA from the. SV40 late promoter and are translatedfrom the first ATG codon in the cloned fragments. Transcripts of clonedDNA are spliced and polyadenylated using the SV40 VPI processingsignals. The leptin gene is cloned into the MCS of pEUK-C1 usingtechniques well known in the art, e.g. J. Sambrook et al., MolecularCloning: A Laboratory Manual (2d ed., 1989), hereby incorporated byreference. The resulting plasmid is delivered to a human or animalindividual after poration of the skin or mucosal membrane according tothe procedure described above in the previous examples.

Example 45

Delivery of Heparin. Heparins are useful therapeutic substances whereinthe maintenance of a basal level equivalent to an intravenous infusionof roughly 1000 to 5000 IU per hour, subcutaneous injections twice dailyof 5000-1000 IU of heparin, or 1500-6000 IU of low molecular weightheparins is a typical clinical dosage. Normally, heparin would not beconsidered a good candidate for a transdermal delivery system because ofits relatively high resistance to crossing the skin due mainly to themolecular weight, 5000 to 30000 Da, of the substance. With themicroporation techniques disclosed herein, a significant flux rate ofheparin was easily achieved when a sufficient quantity of heparin, suchas from a delivery reservoir attached to the skin surface where themicropores were placed, was administered. A heparin solution was appliedto skin porated to a depth of approximately 100 μm, allowing eitherpassive diffusion or coupled with iontophoresis (about 1 mA/cm²) thatwas applied for a sufficient period of time to transport the heparinthrough the micropores into the underlying tissues. Evidence of deliveryof heparin was observed by increased capillary dilation and permeabilityas evidenced by microscopic examination of the in vivo site for both thepassive and iontophoretically enhanced delivery. In addition to showinga significant heparin flux using passive diffusion as the main drivingforce, heparin, being a highly charged compound, is a natural candidatefor the coupling of an electrical field with the micropores to allow foran actively controllable flux rate and higher flux rates than possiblethrough the same number of micropores than is possible with the passivediffusion method. An experiment was conducted wherein a site on thevolar forearm of a healthy male volunteer was prepared by creating amatrix of 36 micropores within a 1 square cm area. A small reservoircontaining a sodium heparin solution and the negative electrode for aniontophoretic system was attached to the site. The positive electrodewas attached to the subject's skin some distance away using a hydrogelelectrode obtained from Iomed, a commercial supplier of iontophoreticsystems. The system was run for ten minutes at 0.2 milliamperes persquare cm. After this period, microscopic examination of the site showeddirect evidence of the delivery of heparin from the vasodilation of thecapillaries and when a suction force was applied to extract a sample ofinterstitial fluid from the micropores, enough red blood cells exitedthe capillaries under this force to tint the collected ISF pink,indicating increased vaso-permeability in the area. Furthermore, whenplaced aside to see if the red cells would clot, no clotting took place,indicating the anticlotting effect of the heparin present in the tissuesat work.

Example 46

Delivery of Insulin: Insulin, like many compounds normally present inthe healthy individual, is a polypeptide which must be maintained inindividuals, such as diabetics who need exogenous insulin, at both abasal level and be given in a pulsatile bolus fashion in response tomeals and the subject's activity levels. Currently this is achieved viasubcutaneous injections of fast acting and slow acting formulations.Because of the molecular weight of insulin, typically ˜6000, it is notable to be delivered at clinically useful levels with traditionaltransdermal or transmucosal methods. However, by opening the microporesthrough the barrier layers of the skin or mucosa, a clear path isprovided allowing the delivery of the insulin into the viable tissueswherein the interstitial fluid present in these tissues will allowdiffusion (including osmotically driven) of the insulin to and into thelymph system and capillary bed, delivering clinically useful amounts. Aconcentrated insulin solution containing 3500 IU/ml of recombinant humaninsulin purchased from Boehringer-Mannhein Co., was applied in areservoir to a crated area of the subject's skin on the volar forearmcovering 4 square cm. The healthy, 44 year old, male, non-diabetic,subject fasted for 14 hours prior to the start of the experiment.Intravenous and finger stick blood samples were drawn periodically priorto and after the delivery phase began and assayed for glucose, insulinand C-peptide. The finger stick blood glucose data showed a significantand rapid depression of the subject's glucose levels after approximately4 hours, dropping from 100 mg/dl at the start to 67 mg/dl over a tenminute cycle and then returning to 100 in an additional ten minutes,hypothesized to be due to the subject's counter-regulatory systemengaging and compensating for the delivered insulin. A repeat of thisprocedure with the addition of ultrasound operating at 44 khz, and 0.2watts/square cm indicated a more rapid delivery of the insulin asevidenced by the subject's glucose levels which dropped from 109 mg/dlto 78 mg/dl less than 30 minutes after the delivery began. As in thecase of example 45, for heparin delivery, a low current iontophoreticsystem can be coupled with the micropores to facilitate a greater fluxrate and provide the ability to modulate this flux rate by varying thecurrent, allowing a delivery on demand type of system to be built.Previous work with insulin has typically shown that relatively highiontophoretic currents are required to overcome the strong barrierproperties of the intact stratum corneum. By porating the stratumcorneum or mucosa, and optionally setting the poration parameters tomake a deeper pore into or through the targeted biological membrane, alower current density is required to produce the desired insulin fluxrates.

Similarly, for uncharged or lower-charged insulin formulations, anactive flux enhancement through the micropores can be effected bycoupling a sonic field or sonophoresis, which may include frequenciesnormally described as ultrasonic, to help push the insulin into thetissues. An additional feature of the sonic field is its ability toenhance the permeability of the various barriers within the viabletissues letting the insulin reach a larger volume of tissue over whichthe desired absorption into the blood stream can take place. Modulatingthe sonic energy has been shown to be very effective in modulating thetotal flux of a compound through the micropores into or through thedeeper tissues, providing a second means of developing a bolus deliverysystem.

The exact pathways of absorption of insulin when given as a subcutaneousinjection are still a subject of some debate. One of the reasons this isstill unclear is the widely varying levels of bio-availabilitydemonstrated within a population, or even the same subject, on aninjection-by-injection basis. One hypothesized pathway is the directabsorption through the capillaries and into the blood stream. A methodfor enhancing this process is to couple electroporation with the surfaceporation, where the electroporation has been specifically optimized towork in the region of the capillary endothelial membranes, creatingtemporarily, a large number of openings to enhance this directabsorption. As with the iontophoresis and sonophoresis describedpreviously, the total voltage amplitude levels of the electroporationsystem required to effect this type of electroporation within thesetissue layers beneath the outer surface are often lower than needed topenetrate through an intact outer surface due to the reduction of thebulk impedance of the outer layer of the biological membrane.

Example 47

Delivery of microparticles: The use of liposomes, lipid complexes,microspheres including nanospheres, PEGellated compounds (compoundscombined with polyethylene glycol) and other microparticles as part of adrug delivery system is well developed for many different specificapplications. In particular, when dealing with a compound which iseasily broken down by the endogenous components in the body's tissuessuch as protease, nuclease, or carbohydrase enzymes in the skin,tissues, the macrophages or other cells present in the blood stream orlymph, increases in bio-availability and/or sustained release canfrequently be realized by utilizing one of these techniques. Currently,once one has applied one of these techniques, the formulation isgenerally delivered via some type of injection. The present invention,by creating micropores through a biological membrane (e.g., the skin ora mucous membrane) and into the body to a selected depth, allows thistype of microparticle to be delivered through the skin or mucosa. Asdescribed in the insulin example above, microporation, electroporation,iontophoresis, sonic energy, enhancers, as well as mechanicalstimulation of the site such as pressure or massage may be combined inany combination to enhance the delivery and/or uptake of a specificformulation. In the case of some engineered microparticles, the poresmay have an optimal to depth designed to bypass certain biologicallyactive zones or place the particle within the zone of choice. For somemicroparticle delivery systems, the energy incident upon the particlesafter they have been delivered into or through the tissues beneath thesurface may be used to trigger the accelerated release of the activecompound, thereby allowing the external control of the flux rate of thetherapeutic substance.

Example 48

Microparticles for implantable analyte monitoring: Another applicationof microparticles is to deliver a particle not as a therapeutic agentbut as a carrier of a probe compound which could be interrogatednon-invasively, for example, via electro-magnetic radiation from anexternal reader system to obtain information regarding the levels of aspecific analyte in the body. One example is to incorporate in a porousmicrosphere a glucose specific fluorophore compound which, depending onthe levels of glucose present in the surrounding tissues, would alterits fluorescent response in either amplitude, wavelength, or fluorescentlifetime. If the fluorophore was designed to be active with anexcitation wavelength ranging from 700 nm to 1500 nm, a low costinfrared light source such as an LED or laser diode could be used tostimulate its fluorescent response, which would similarly be in thisrange of from 700 nm to 1500 nm. At these wavelengths, the skin andmucosal tissues absorb very little and would therefore allow a simplesystem to be built along these lines.

Glucose is one candidate analyte, for which experimental lifetimefluorescence probes have been developed and incorporated intosubcutaneously inserted polymer implants which have been successfullyinterrogated through the skin with optical stimulation and detectionmethods. It would merely require the reformulation of these experimentalimplants into suitably sized microparticles to allow the delivery intoor through the viable tissue layers via the micropores. However, anyanalyte could be targeted, and the method of interrogating the deliveredmicroparticles could be via magnetic or electric field rather thanoptical energy.

Example 49 Delivery of a Vaccine

A bacterial, viral, toxoid or mixed vaccine is prepared as a solid,liquid, suspension, or gel as required. This formulation could includeany one or combination of peptides, proteins, carbohydrates, DNA, RNA,entire microorganisms, adjuvants, carriers and the like. A selected siteof an individual is porated (skin or mucous membrane) according to theprocedures described above in Example 45 and the vaccine is applied tothe porated site. The depth of the micropores may depend on the type ofvaccine delivered. This delivery can be aided with electroporation,iontophoresis, magnetic or sonic energy, enhancers, as well asmechanical stimulation of the site such as pressure or massage accordingto the procedures described above and/or use electroporation,iontophoresis, magnetic or sonic energy, enhancers, as well asmechanical stimulation of the site such as pressure or massage toenhance cellular uptake. Additional or reinforcing doses can bedelivered in the same manner to achieve immunization of the individual.

Example 50

Delivery of Testosterone: A commercially available testosterone patch,the Androderm^(R)patch from TheraTech, Inc., was used in a set ofexperiments to evaluate the benefits of microporation as it applies tothe delivery of this permeant. A hypergonadic male subject went offAndroderm therapy for two days, after which a series of venous bloodsamples were drawn during the subsequent 24 hour period to establishthis subject's baseline levels of testosterone. Two 2.5 mg Androdermpatches were then installed as recommended by the manufacturer and asimilar set of venous blood sample were drawn to measure thetestosterone levels when the only transdermal flux enhancement methodbeing used was the chemical permeation enhancers contained in the patch.After two more days of a washout period, two 2.5 Androderm patches werethen similarly installed, but prior to the installation, the skinsurface at the target sites was porated with 72 micropores per site,each pore measuring approximately 80 μm in width and 300 μm in lengthand extending to a depth of 80 to 120 μm. For the porated delivery phasea similar set of venous blood sample were drawn to measure thetestosterone. The data from all three of these twenty four hour periodsis shown in the FIG. 35 titled ‘Effects of Microporation on TransdermalTestosterone Delivery’. A noteworthy feature of these data is that whenthe microporations are present, the testosterone levels in the subjectsblood elevate much more rapidly, essentially preceding the rising edgeof the un-porated cycle by more than four hours. Looking at the slope ofand area under the curve we can calculate that more than a three-foldflux rate enhancement took place due to the microporations during thefirst four hours.

Example 51

Delivery of Alprostadil: Alprostadil, or PGE1, is a prostaglandin usedtherapeutically to treat male erectile dysfunction via it's vasodilatorbehavior. The standard delivery mode for this drug is a direct injectioninto the base of the penis or via a suppository inserted into theurethra. A set of experiments were conducted with two healthy malevolunteers. Each subject had a site of 1 square cm on the base of thepenis shaft prepared by porating 12 to 36 micropores on this area, withthe thermal poration parameters set to create pores roughly 100 micronsdeep as measured from the surface of the skin. A concentrated solutionof alprostadil was placed in a small reservoir patch placed on theporation site, an ultrasonic transducer was then placed on the top ofthe reservoir and activated and the subject's erectile and otherclinical responses were recorded on video tape. Both subjects developeda significant amount of engorgement of the penis, estimated as achieving70% of more of a full erection at the dose applied. In addition, a malarflush response to the systemic levels of the drug delivered wasobserved. Over a 30 to 60 minute delivery period, both subjectsdeveloped a profound malar flush extending from the face, neck, chestand arms. Both the erectile response and the malar flush provideevidence of the delivery of a clinically active amount of the drug, awell know vasodilator.

Example 52

Delivery of Interferon: Interferons are proteins of approximately17-22,000 molecular weight, that are administered clinically to treat avariety of disease states, such as viral infections (e.g., hepatitis Band C), immune diseases (such as multiple sclerosis), and cancers (e.g.,hairy cell leukemia). Due to their protein nature, interferons mustcurrently be administered by injection, as they cannot be given orallyand are too large for traditional transdermal or transmucosal deliverymethods. To demonstrate delivery of an interferon via the microporationtechnique, a 100 microliter aliquot of alpha-interferon solutioncontaining interferon with a specific activity of 100 millioninternational units of interferon per mg dissolved in 1 ml of deliverysolution, is applied to a 1 square cm area of porated skin, porated to adepth of 150-180 μm, thus falling short of the capillary bed, on thethigh of a healthy human subject. Trials are run using either purelypassive diffusion and with the application of sonic energy to the regionat sufficient amplitude, frequency, and modulation thereof to acceleratethe migration of the interferon through pores into or through theunderlying tissues without causing deleterious heating of the interferonsolution. Venous blood draws are taken at various time intervals forboth trials, and are assayed for interferon levels usingradio-immunoassay and bioassay. Interferon is detected in the serum overthe 4 hour time period monitored. The interferon levels for thesonically enhanced delivery experiment are detected sooner than for thepassive experiment. In another experiment, the interferon isadministered in dry powder form directly to the micropores in theporated area of the skin. Interferon is detected in the serum using thesame techniques as described above. In another test, the interferonsolution is applied in a gel with or without a backing film to theporated tissue of the buccal mucosa. Venous blood is drawn and assayedfor interferon levels. Interferon is detected in the serum over the 3hour time period monitored. In another experiment, the interferon isincorporated into a tablet containing a bio-erodable matrix, with amucoadherent polymer matrix that provided contact of the tablet over thearea of buccal mucosa that is porated. Interferon is detected in theserum using the same techniques as described above.

Example 53

Delivery of morphine: A solution of morphine is applied to a poratedarea on the volar forearm of the human subjects. A positive pressuregradient is used to provide a basal delivery rate of the morphine intothe body, as determined by assay of venous blood draws at appropriatetime intervals for the presence of morphine. A basal level of morphineof approximately 3-6 ng/ml is achieved. Upon demand, an additionalpressure bolus is applied to result in a spike in the delivery of themorphine. The additional pressure bolus is achieved in one test by useof ultrasound; or in another experiment by the use of a pressure spike.This type of delivery, in which a basal level of the morphine iscontinuously applied, with spikes in morphine delivery periodically upondemand, is useful in treating chronic and breakthrough pain.

Example 54

Delivery of a disease resistant DNA into a plant: The seeds of aselected corn plant are microporated. The seeds are placed in a solutionof a permeant formulation containing DNA that encodes disease resistanceproteins. Sonic energy is used, optionally, to enhance the delivery ofthe DNA into the corn seeds. The seeds are germinated and grown tomaturity. The resulting seeds of the mature corn plants now carry thedisease resistant gene.

Example 55

Delivery of DNA into a plant: The seeds of a sugar beet aremicroporated. The seeds are placed in a solution of a permeantformulation containing DNA that encodes human growth hormone.Electroporation, iontophoresis, sonic energy, enhancers, as well asmechanical stimulation of the site such as pressure may be used toenhance the delivery of the DNA into the seeds. The seeds are germinatedand grown to maturity. The resulting mature beet plant can now beharvested and the human growth hormone extracted for subsequentpurification and clinical use.

Example 56

An experiment was conducted wherein fluorescent dextran particles, MWapproximately 10,000 Daltons, were applied in an aqueous solution bymeans of a reservoir patch over a one square cm of skin on the volarforearm of a human subject where 36 micropores extending approximately80 μm in depth were formed. The reservoir patch was left in place for 5minutes. The porated site and surrounding area were imaged with afluorescent video microscope to evaluate the penetration of the permeantinto the tissue. The fluorescence showed that within 5 minutessignificant permeation of dextran occurred more than 2 mm away from thenearest micropore. The video assay system used 10 minutes later showedfurther diffusion so that the fluorescent flush extended 10 mm from thepores. This experiment gives clear evidence that this technique allowsdelivery of permeants with molecular weights of 10,000.

1-73. (canceled)
 74. An apparatus for delivering a formulation into anorganism comprising: a supply of a dry powder formulation; and a heatconducting element, which, when put in substantial physical contact witha selected area of a biological membrane of the organism, is capable ofporating the biological membrane at the selected area to form at leastone micropore 1-1000 μm in diameter, by delivering sufficient energy tothe selected area such that the temperature of tissue-bound water andother vaporizable substances in the selected area is elevated above thevaporization point of the water and other vaporizable substances,thereby removing the biological membrane in the selected area, andwherein the apparatus enables the dry powder formulation, when the drypowder formulation is put in contact with the selected area, to be takenup through the micropore into the organism, wherein the apparatusdelivers the dry powder formulation into the organism.
 75. The apparatusof claim 74, wherein the dry powder formulation comprises a peptide(s),protein(s), vaccine antigen, DNA or RNA.
 76. The apparatus of claim 74,wherein the dry powder formulation comprises adenovirus.
 77. Theapparatus of claim 74, wherein the dry powder formulation comprisesmicroparticles.
 78. The apparatus of claim 77, wherein saidmicroparticles comprise a bioactive agent(s).
 79. The apparatus of claim78, wherein said bioactive agent(s) is selected from the groupconsisting of peptide(s), protein(s), vaccine antigen(s), DNA or RNA.80. The apparatus of claim 79, wherein said DNA or RNA is naked,fragmented, encapsulated or coupled to another agent.
 81. An apparatusfor delivering a bioactive agent into an organism comprising: a heatconducting element, which, when put in substantial physical contact witha selected area of a biological membrane of the organism, is capable ofporating the biological membrane at the selected area to form at leastone micropore 1-1000 μm in diameter, by delivering sufficient energy tothe selected area such that the temperature of tissue-bound water andother vaporizable substances in the selected area is elevated above thevaporization point of the water and other vaporizable substances,thereby removing the biological membrane in the selected area, andwherein the apparatus enables the bioactive agent, when the bioactiveagent is put in contact with the selected area, to be taken up throughthe micropore into the organism, wherein said bioactive agent is put incontact with the selected area in a form selected from the groupconsisting of a tablet and a bio-erodable matrix, wherein saidbio-erodable matrix is fabricated in a manner to allow the bioactiveagent to be released into the organism via the micropore, wherein theapparatus delivers the bioactive agent into the organism.
 82. A systemfor stimulating an immune response in an organism, comprising: a supplyof a permeant; and a heat conducting element, which, when put insubstantial physical contact with a selected area of a biologicalmembrane of the organism, is capable of porating the biological membraneat the selected area to form at least one micropore 1-1000 μm indiameter and at a depth coincident with increased concentration oflangerhans cells, by delivering sufficient energy to the selected areasuch that the temperature of tissue-bound water and other vaporizablesubstances in the selected area is elevated above the vaporization pointof the water and other vaporizable substances, thereby removing thebiological membrane in the selected area, and wherein the system enablesthe permeant, when the permeant is put in contact with the selectedarea, to be taken up through the micropore into the organism, whereinthe system stimulates the immune response in the organism.
 83. Thesystem of claim 82, wherein the depth of the micropore is 180 microns to250 microns.
 84. The system of claim 82, wherein the organism is ananimal.
 85. The system of claim 84, wherein the animal is a human. 86.The system of claim 82, wherein the permeant is a vaccine.
 87. Thesystem of claim 86, wherein the vaccine comprises DNA or RNA.
 88. Thesystem of claim 82, wherein the permeant is introduced into theepidermis via formed micropores at a depth coincident with increasedconcentration of langerhans cells.
 89. The system of claim 82, whereinthe surface area of the selected area of the biological membrane isgreater than the total area of the micropores.
 90. A system fordelivering a formulation into an organism comprising: a supply of a drypowder formulation; and a heat conducting element, which, when put insubstantial physical contact with a selected area of a biologicalmembrane of the organism, is capable of porating the biological membraneat the selected area to form at least one micropore 1-1000 μm indiameter, by delivering sufficient energy to the selected area such thatthe temperature of tissue-bound water and other vaporizable substancesin the selected area is elevated above the vaporization point of thewater and other vaporizable substances, thereby removing the biologicalmembrane in the selected area, and wherein the system enables the drypowder formulation, when the dry powder formulation is put in contactwith the selected area, to be taken up through the micropore into theorganism, wherein the system delivers the dry powder formulation intothe organism.
 91. The system of claim 90, wherein the dry powderformulation comprises a peptide(s), protein(s), vaccine antigen(s), DNAor RNA.
 92. The system of claim 90, wherein the dry powder formulationcomprises microparticles.
 93. The system of claim 92, wherein saidmicroparticles comprise a bioactive agent(s).
 94. The system of claim93, wherein said bioactive agent(s) is selected from the groupconsisting of peptide(s), protein(s), vaccine antigen(s), DNA or RNA.95. The system of claim 94, wherein said DNA or RNA is naked,fragmented, encapsulated or coupled to another agent.